Codon Usage Selection Can Bias Estimation of the Fraction of Adaptive Amino Acid Fixations.

PubMed

Matsumoto, Tomotaka; John, Anoop; Baeza-Centurion, Pablo; Li, Boyang; Akashi, Hiroshi

2016-06-01

A growing number of molecular evolutionary studies are estimating the proportion of adaptive amino acid substitutions (α) from comparisons of ratios of polymorphic and fixed DNA mutations. Here, we examine how violations of two of the model assumptions, neutral evolution of synonymous mutations and stationary base composition, affect α estimation. We simulated the evolution of coding sequences assuming weak selection on synonymous codon usage bias and neutral protein evolution, α = 0. We show that weak selection on synonymous mutations can give polymorphism/divergence ratios that yield α-hat (estimated α) considerably larger than its true value. Nonstationary evolution (changes in population size, selection, or mutation) can exacerbate such biases or, in some scenarios, give biases in the opposite direction, α-hat < α. These results demonstrate that two factors that appear to be prevalent among taxa, weak selection on synonymous mutations and non-steady-state nucleotide composition, should be considered when estimating α. Estimates of the proportion of adaptive amino acid fixations from large-scale analyses of Drosophila melanogaster polymorphism and divergence data are positively correlated with codon usage bias. Such patterns are consistent with α-hat inflation from weak selection on synonymous mutations and/or mutational changes within the examined gene trees. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Homozygous MYH7 R1820W mutation results in recessive myosin storage myopathy: scapuloperoneal and respiratory weakness with dilated cardiomyopathy.

PubMed

Yüceyar, Nur; Ayhan, Özgecan; Karasoy, Hatice; Tolun, Aslıhan

2015-04-01

Myosin storage myopathy (MSM) is a protein aggregate myopathy caused by the accumulation of myosin in muscle fibres and results from MYH7 mutation. Although MYH7 mutation is also an established cause of variable cardiomyopathy with or without skeletal myopathy, cardiomyopathy with MSM is a rare combination. Here, we update the clinical findings in the two brothers that we previously reported as having recessively inherited MSM characterized by scapuloperoneal distribution of weakness and typical hyaline-like bodies in type 1 muscle fibres. One of the patients, weak from childhood but not severely symptomatic until 28 years of age, had an unusual combination of MSM, severe dilated cardiomyopathy, and respiratory impairment at the age of 44 years. We identified homozygous missense mutation c.5458C>T (p.R1820W) in exon 37 in these patients as the second recessive MYH7 mutation reported to date. Copyright © 2015 Elsevier B.V. All rights reserved.

Loss of ATM kinase activity leads to embryonic lethality in mice.

PubMed

Daniel, Jeremy A; Pellegrini, Manuela; Lee, Baeck-Seung; Guo, Zhi; Filsuf, Darius; Belkina, Natalya V; You, Zhongsheng; Paull, Tanya T; Sleckman, Barry P; Feigenbaum, Lionel; Nussenzweig, André

2012-08-06

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

An experimental SMI adaptive antenna array for weak interfering signals

NASA Technical Reports Server (NTRS)

Dilsavor, R. L.; Gupta, I. J.

1989-01-01

A modified sample matrix inversion (SMI) algorithm designed to increase the suppression of weak interference is implemented on an existing experimental array system. The algorithm itself is fully described as are a number of issues concerning its implementation and evaluation, such as sample scaling, snapshot formation, weight normalization, power calculation, and system calibration. Several experiments show that the steady state performance (i.e., many snapshots are used to calculate the array weights) of the experimental system compares favorably with its theoretical performance. It is demonstrated that standard SMI does not yield adequate suppression of weak interference. Modified SMI is then used to experimentally increase this suppression by as much as 13dB.

Mutation-selection equilibrium in games with multiple strategies.

PubMed

Antal, Tibor; Traulsen, Arne; Ohtsuki, Hisashi; Tarnita, Corina E; Nowak, Martin A

2009-06-21

In evolutionary games the fitness of individuals is not constant but depends on the relative abundance of the various strategies in the population. Here we study general games among n strategies in populations of large but finite size. We explore stochastic evolutionary dynamics under weak selection, but for any mutation rate. We analyze the frequency dependent Moran process in well-mixed populations, but almost identical results are found for the Wright-Fisher and Pairwise Comparison processes. Surprisingly simple conditions specify whether a strategy is more abundant on average than 1/n, or than another strategy, in the mutation-selection equilibrium. We find one condition that holds for low mutation rate and another condition that holds for high mutation rate. A linear combination of these two conditions holds for any mutation rate. Our results allow a complete characterization of nxn games in the limit of weak selection.

A novel missense HGD gene mutation, K57N, in a patient with alkaptonuria.

PubMed

Grasko, Jonathan M; Hooper, Amanda J; Brown, Jeffrey W; McKnight, C James; Burnett, John R

2009-05-01

Alkaptonuria is a rare recessive disorder of phenylalanine/tyrosine metabolism due to a defect in the enzyme homogentisate 1,2-dioxygenase (HGD) caused by mutations in the HGD gene. We report the case of a 38 year-old male with known alkaptonuria who was referred to an adult metabolic clinic after initially presenting to an emergency department with renal colic and subsequently passing black ureteric calculi. He complained of severe debilitating lower back pain, worsening over the last few years. A CT scan revealed marked degenerative changes and severe narrowing of the disc spaces along the entire lumbar spine. Sequencing of the HGD gene revealed that he was a compound heterozygote for a previously described missense mutation in exon 13 (G360R) and a novel missense mutation in exon 3 (K57N). Lys(57) is conserved among species and mutation of this residue is predicted to affect HGD protein function by interfering with substrate traffic at the active site. In summary, we describe an alkaptonuric patient and report a novel missense HGD mutation, K57N.

Selective Strolls: Fixation and Extinction in Diploids Are Slower for Weakly Selected Mutations Than for Neutral Ones.

PubMed

Mafessoni, Fabrizio; Lachmann, Michael

2015-12-01

In finite populations, an allele disappears or reaches fixation due to two main forces, selection and drift. Selection is generally thought to accelerate the process: a selected mutation will reach fixation faster than a neutral one, and a disadvantageous one will quickly disappear from the population. We show that even in simple diploid populations, this is often not true. Dominance and recessivity unexpectedly slow down the evolutionary process for weakly selected alleles. In particular, slightly advantageous dominant and mildly deleterious recessive mutations reach fixation slightly more slowly than neutral ones (at most 5%). This phenomenon determines genetic signatures opposite to those expected under strong selection, such as increased instead of decreased genetic diversity around the selected site. Furthermore, we characterize a new phenomenon: mildly deleterious recessive alleles, thought to represent a wide fraction of newly arising mutations, on average survive in a population slightly longer than neutral ones, before getting lost. Consequently, these mutations are on average slightly older than neutral ones, in contrast with previous expectations. Furthermore, they slightly increase the amount of weakly deleterious polymorphisms, as a consequence of the longer unconditional sojourn times compared to neutral mutations. Copyright © 2015 by the Genetics Society of America.

Adaptive antenna arrays for satellite communications: Design and testing

NASA Technical Reports Server (NTRS)

Gupta, I. J.; Swarner, W. G.; Walton, E. K.

1985-01-01

When two separate antennas are used with each feedback loop to decorrelate noise, the antennas should be located such that the phase of the interfering signal in the two antennas is the same while the noise in them is uncorrelated. Thus, the antenna patterns and spatial distribution of the auxiliary antennas are quite important and should be carefully selected. The selection and spatial distribution of auxiliary elements is discussed when the main antenna is a center fed reflector antenna. It is shown that offset feeds of the reflector antenna can be used as auxiliary elements of an adaptive array to suppress weak interfering signals. An experimental system is designed to verify the theoretical analysis. The details of the experimental systems are presented.

Performance of a modified feedback loop adaptive array with TVRO satellite signals

NASA Technical Reports Server (NTRS)

Steadman, Karl N.; Gupta, Inder J.; Walton, Eric K.

1990-01-01

Performance of an experimental adaptive antenna array system is evaluated using television receive-only (TVRO) satellite signals. The experimental system is a sidelobe canceller with two auxiliary channels. Modified feedback loops are used to enhance the suppression of weak interfering signals. The modified feedback loops used two spatialy separated antennas, each with an individual amplifier for each auxiliary channel. Thus, the experimental system uses five antenna elements. Instead of using five separate antennas, a reflector antenna with multiple feeds is used to receive signals from various TVRO satellites. The details of the earth station are given. It is shown that the experimental system can null up to two signals originating from interfering TVRO satellites while receiving the signals from a desired TVRO satellite.

Recessive Mutations in ACPT, Encoding Testicular Acid Phosphatase, Cause Hypoplastic Amelogenesis Imperfecta.

PubMed

Seymen, Figen; Kim, Youn Jung; Lee, Ye Ji; Kang, Jenny; Kim, Tak-Heun; Choi, Hwajung; Koruyucu, Mine; Kasimoglu, Yelda; Tuna, Elif Bahar; Gencay, Koray; Shin, Teo Jeon; Hyun, Hong-Keun; Kim, Young-Jae; Lee, Sang-Hoon; Lee, Zang Hee; Zhang, Hong; Hu, Jan C-C; Simmer, James P; Cho, Eui-Sic; Kim, Jung-Wook

2016-11-03

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders affecting tooth enamel. The affected enamel can be hypoplastic and/or hypomineralized. In this study, we identified ACPT (testicular acid phosphatase) biallelic mutations causing non-syndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta (AI) in individuals from six apparently unrelated Turkish families. Families 1, 4, and 5 were affected by the homozygous ACPT mutation c.713C>T (p.Ser238Leu), family 2 by the homozygous ACPT mutation c.331C>T (p.Arg111Cys), family 3 by the homozygous ACPT mutation c.226C>T (p.Arg76Cys), and family 6 by the compound heterozygous ACPT mutations c.382G>C (p.Ala128Pro) and 397G>A (p.Glu133Lys). Analysis of the ACPT crystal structure suggests that these mutations damaged the activity of ACPT by altering the sizes and charges of key amino acid side chains, limiting accessibility of the catalytic core, and interfering with homodimerization. Immunohistochemical analysis confirmed localization of ACPT in secretory-stage ameloblasts. The study results provide evidence for the crucial function of ACPT during amelogenesis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

How Large Asexual Populations Adapt

NASA Astrophysics Data System (ADS)

Desai, Michael

2007-03-01

We often think of beneficial mutations as being rare, and of adaptation as a sequence of selected substitutions: a beneficial mutation occurs, spreads through a population in a selective sweep, then later another beneficial mutation occurs, and so on. This simple picture is the basis for much of our intuition about adaptive evolution, and underlies a number of practical techniques for analyzing sequence data. Yet many large and mostly asexual populations -- including a wide variety of unicellular organisms and viruses -- live in a very different world. In these populations, beneficial mutations are common, and frequently interfere or cooperate with one another as they all attempt to sweep simultaneously. This radically changes the way these populations adapt: rather than an orderly sequence of selective sweeps, evolution is a constant swarm of competing and interfering mutations. I will describe some aspects of these dynamics, including why large asexual populations cannot evolve very quickly and the character of the diversity they maintain. I will explain how this changes our expectations of sequence data, how sex can help a population adapt, and the potential role of ``mutator'' phenotypes with abnormally high mutation rates. Finally, I will discuss comparisons of these predictions with evolution experiments in laboratory yeast populations.

Exome sequencing identifies a novel SMCHD1 mutation in facioscapulohumeral muscular dystrophy 2.

PubMed

Mitsuhashi, Satomi; Boyden, Steven E; Estrella, Elicia A; Jones, Takako I; Rahimov, Fedik; Yu, Timothy W; Darras, Basil T; Amato, Anthony A; Folkerth, Rebecca D; Jones, Peter L; Kunkel, Louis M; Kang, Peter B

2013-12-01

FSHD2 is a rare form of facioscapulohumeral muscular dystrophy (FSHD) characterized by the absence of a contraction in the D4Z4 macrosatellite repeat region on chromosome 4q35 that is the hallmark of FSHD1. However, hypomethylation of this region is common to both subtypes. Recently, mutations in SMCHD1 combined with a permissive 4q35 allele were reported to cause FSHD2. We identified a novel p.Lys275del SMCHD1 mutation in a family affected with FSHD2 using whole-exome sequencing and linkage analysis. This mutation alters a highly conserved amino acid in the ATPase domain of SMCHD1. Subject III-11 is a male who developed asymmetrical muscle weakness characteristic of FSHD at 13 years. Physical examination revealed marked bilateral atrophy at biceps brachii, bilateral scapular winging, some asymmetrical weakness at tibialis anterior and peroneal muscles, and mild lower facial weakness. Biopsy of biceps brachii in subject II-5, the father of III-11, demonstrated lobulated fibers and dystrophic changes. Endomysial and perivascular inflammation was found, which has been reported in FSHD1 but not FSHD2. Given the previous report of SMCHD1 mutations in FSHD2 and the clinical presentations consistent with the FSHD phenotype, we conclude that the SMCHD1 mutation is the likely cause of the disease in this family. Copyright © 2013 Elsevier B.V. All rights reserved.

FUS GENE MUTATIONS IN FAMILIAL AND SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

PubMed Central

Rademakers, Rosa; Stewart, Heather; DeJesus-Hernandez, Mariely; Krieger, Charles; Graff-Radford, Neill; Fabros, Marife; Briemberg, Hannah; Cashman, Neil; Eisen, Andrew; Mackenzie, Ian R. A.

2010-01-01

Introduction Mutations in the fused in sarcoma (FUS) gene have recently been found to cause familial amyotrophic lateral sclerosis (FALS). Methods We screened FUS in a cohort of 200 ALS patients [32 FALS and 168 sporadic ALS (SALS)]. Results In one FALS proband, we identified a mutation (p.R521C) that was also present in her affected daughter. Their clinical phenotype was remarkably similar and atypical of classic ALS, with symmetric proximal pelvic and pectoral weakness. Distal weakness and upper motor neuron features only developed late. Neuropathological examination demonstrated FUS-immunoreactive neuronal and glial inclusions in the spinal cord and many extramotor regions, but no TDP-43 pathology. We also identified a novel mutation (p.G187S) in one SALS patient. Overall, FUS mutations accounted for 3% of our non-SOD1, non-TARDBP FALS cases and 0.6% of SALS. Discussion This study demonstrates that the phenotype with FUS mutations extends beyond classical ALS. It suggests there are specific clinicogenetic correlations and provides the first detailed neuropathological description. PMID:20544928

Extraction of a Weak Co-Channel Interfering Communication Signal Using Complex Independent Component Analysis

DTIC Science & Technology

2013-06-01

zarzoso/ biblio /tnn10.pdf"> % "Robust independent component analysis by iterative maximization</a> % <a href = "http://www.i3s.unice.fr/~zarzoso... biblio /tnn10.pdf"> % of the kurtosis contrast with algebraic optimal step size"</a>, % IEEE Transactions on Neural Networks, vol. 21, no. 2, % pp

Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

PubMed

Kaltenbach, Miriam; Emond, Stephane; Hollfelder, Florian; Tokuriki, Nobuhiko

2016-10-01

The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.

Agrin mutations lead to a congenital myasthenic syndrome with distal muscle weakness and atrophy.

PubMed

Nicole, Sophie; Chaouch, Amina; Torbergsen, Torberg; Bauché, Stéphanie; de Bruyckere, Elodie; Fontenille, Marie-Joséphine; Horn, Morten A; van Ghelue, Marijke; Løseth, Sissel; Issop, Yasmin; Cox, Daniel; Müller, Juliane S; Evangelista, Teresinha; Stålberg, Erik; Ioos, Christine; Barois, Annie; Brochier, Guy; Sternberg, Damien; Fournier, Emmanuel; Hantaï, Daniel; Abicht, Angela; Dusl, Marina; Laval, Steven H; Griffin, Helen; Eymard, Bruno; Lochmüller, Hanns

2014-09-01

Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of rare diseases resulting from impaired neuromuscular transmission. Their clinical hallmark is fatigable muscle weakness associated with a decremental muscle response to repetitive nerve stimulation and frequently related to postsynaptic defects. Distal myopathies form another clinically and genetically heterogeneous group of primary muscle disorders where weakness and atrophy are restricted to distal muscles, at least initially. In both congenital myasthenic syndromes and distal myopathies, a significant number of patients remain genetically undiagnosed. Here, we report five patients from three unrelated families with a strikingly homogenous clinical entity combining congenital myasthenia with distal muscle weakness and atrophy reminiscent of a distal myopathy. MRI and neurophysiological studies were compatible with mild myopathy restricted to distal limb muscles, but decrement (up to 72%) in response to 3 Hz repetitive nerve stimulation pointed towards a neuromuscular transmission defect. Post-exercise increment (up to 285%) was observed in the distal limb muscles in all cases suggesting presynaptic congenital myasthenic syndrome. Immunofluorescence and ultrastructural analyses of muscle end-plate regions showed synaptic remodelling with denervation-reinnervation events. We performed whole-exome sequencing in two kinships and Sanger sequencing in one isolated case and identified five new recessive mutations in the gene encoding agrin. This synaptic proteoglycan with critical function at the neuromuscular junction was previously found mutated in more typical forms of congenital myasthenic syndrome. In our patients, we found two missense mutations residing in the N-terminal agrin domain, which reduced acetylcholine receptors clustering activity of agrin in vitro. Our findings expand the spectrum of congenital myasthenic syndromes due to agrin mutations and show an unexpected correlation between the mutated gene and the associated phenotype. This provides a good rationale for examining patients with apparent distal myopathy for a neuromuscular transmission disorder and agrin mutations. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

A novel mutation in PNPLA2 leading to neutral lipid storage disease with myopathy.

PubMed

Ash, Daniel B; Papadimitriou, Dimitra; Hays, Arthur P; Dimauro, Salvatore; Hirano, Michio

2012-09-01

Mutations in PNPLA2, a gene encoding adipose triglyceride lipase, lead to neutral lipid storage disease with myopathy. To report the clinical and molecular features of a case of neutral lipid storage disease with myopathy resulting from a novel mutation in PNPLA2. Case report. University hospital. A 65-year-old man with progressive muscle weakness and high serum creatine kinase levels. Direct sequencing of the PNPLA2 gene. Identification of a novel homozygous mutation in the patient's PNPLA2 gene confirmed the suspected diagnosis of neutral lipid storage disease with myopathy. Screening of the PNPLA2 gene should be considered for patients presenting with high levels of creatine kinase, progressive muscle weakness, and systemic lipid accumulation. The presence of Jordans anomaly can be a strong diagnostic clue.

BAG3 myofibrillar myopathy presenting with cardiomyopathy.

PubMed

Konersman, Chamindra G; Bordini, Brett J; Scharer, Gunter; Lawlor, Michael W; Zangwill, Steven; Southern, James F; Amos, Louella; Geddes, Gabrielle C; Kliegman, Robert; Collins, Michael P

2015-05-01

Myofibrillar myopathies (MFMs) are a heterogeneous group of neuromuscular disorders distinguished by the pathological hallmark of myofibrillar dissolution. Most patients present in adulthood, but mutations in several genes including BCL2-associated athanogene 3 (BAG3) cause predominantly childhood-onset disease. BAG3-related MFM is particularly severe, featuring weakness, cardiomyopathy, neuropathy, and early lethality. While prior cases reported either neuromuscular weakness or concurrent weakness and cardiomyopathy at onset, we describe the first case in which cardiomyopathy and cardiac transplantation (age eight) preceded neuromuscular weakness by several years (age 12). The phenotype comprised distal weakness and severe sensorimotor neuropathy. Nerve biopsy was primarily axonal with secondary demyelinating/remyelinating changes without "giant axons." Muscle biopsy showed extensive neuropathic changes that made myopathic changes difficult to interpret. Similar to previous cases, a p.Pro209Leu mutation in exon 3 of BAG3 was found. This case underlines the importance of evaluating for MFMs in patients with combined neuromuscular weakness and cardiomyopathy. Copyright © 2015 Elsevier B.V. All rights reserved.

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or ‘classical’ congenital myopathy

PubMed Central

Zaharieva, Irina T.; Thor, Michael G.; Oates, Emily C.; van Karnebeek, Clara; Hendson, Glenda; Blom, Eveline; Witting, Nanna; Rasmussen, Magnhild; Gabbett, Michael T.; Ravenscroft, Gianina; Sframeli, Maria; Suetterlin, Karen; Sarkozy, Anna; D’Argenzio, Luigi; Hartley, Louise; Matthews, Emma; Pitt, Matthew; Vissing, John; Ballegaard, Martin; Krarup, Christian; Slørdahl, Andreas; Halvorsen, Hanne; Ye, Xin Cynthia; Zhang, Lin-Hua; Løkken, Nicoline; Werlauff, Ulla; Abdelsayed, Mena; Davis, Mark R.; Feng, Lucy; Phadke, Rahul; Sewry, Caroline A.; Morgan, Jennifer E.; Laing, Nigel G.; Vallance, Hilary; Ruben, Peter; Hanna, Michael G.; Lewis, Suzanne; Kamsteeg, Erik-Jan; Männikkö, Roope

2016-01-01

Abstract See Cannon (doi: 10.1093/brain/awv400 ) for a scientific commentary on this article. Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Na v 1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero - or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Na v 1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Na v 1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy. PMID:26700687

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy.

PubMed

Zaharieva, Irina T; Thor, Michael G; Oates, Emily C; van Karnebeek, Clara; Hendson, Glenda; Blom, Eveline; Witting, Nanna; Rasmussen, Magnhild; Gabbett, Michael T; Ravenscroft, Gianina; Sframeli, Maria; Suetterlin, Karen; Sarkozy, Anna; D'Argenzio, Luigi; Hartley, Louise; Matthews, Emma; Pitt, Matthew; Vissing, John; Ballegaard, Martin; Krarup, Christian; Slørdahl, Andreas; Halvorsen, Hanne; Ye, Xin Cynthia; Zhang, Lin-Hua; Løkken, Nicoline; Werlauff, Ulla; Abdelsayed, Mena; Davis, Mark R; Feng, Lucy; Phadke, Rahul; Sewry, Caroline A; Morgan, Jennifer E; Laing, Nigel G; Vallance, Hilary; Ruben, Peter; Hanna, Michael G; Lewis, Suzanne; Kamsteeg, Erik-Jan; Männikkö, Roope; Muntoni, Francesco

2016-03-01

Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.

Performance of a modified feedback loop adaptive array with TVRO satellite signals

NASA Technical Reports Server (NTRS)

Steadman, K.; Gupta, I. J.; Walton, E. K.

1990-01-01

The performance of an experimental adaptive antenna array system is evaluated using television-receive-only (TVRO) satellite signals. The experimental system is a sidelobe canceler with two auxiliary channels. Modified feedback loops are used to enhance the suppression of weak interfering signals. The modified feedback loops use two spatially separate antennas, each with an individual amplifier for each auxiliary channel. Thus, the experimental system uses five antenna elements. Instead of using five separate antennas, a reflector antenna with multiple feeds is used to receive signals from various TVRO satellites. The details of the earth station are given. It is shown that the experimental system can null up to two signals originating from interfering TVRO satellites while receiving the signals from a desired TVRO satellite.

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

NASA Technical Reports Server (NTRS)

Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

2005-01-01

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Recovery of Spectrally Overlapping QPSK Signals Using a Nonlinear Optoelectronic Filter

DTIC Science & Technology

2017-03-19

Spectrally Overlapping QPSK Signals Using a Nonlinear Optoelectronic Filter William Loh, Siva Yegnanarayanan, Kenneth E. Kolodziej, and Paul...recovery of a weak QPSK signal buried 35-dB beneath an interfering QPSK signal having an overlapping spectrum. This nonlinear optoelectronic filter ...from increased detection sensitivity. Here, we demonstrate an optoelectronic filter that enables the detection of a desired signal hidden beneath a

Extraction of a Weak Co-Channel Interfering Communication Signal Using Adaptive Filtering

DTIC Science & Technology

2015-03-01

is unlimited THIS PAGE INTENTIONALLY LEFT BLANK REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704–0188 Public reporting burden for this collection...and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other...aspect of this collection of information, including suggestions for reducing this burden to Washington headquarters Services, Directorate for

MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots[W

PubMed Central

Xia, Rui; Meyers, Blake C.; Liu, Zhongchi; Beers, Eric P.; Ye, Songqing; Liu, Zongrang

2013-01-01

Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families. PMID:23695981

Germline hypomorphic CARD11 mutations in severe atopic disease

PubMed Central

Ma, Chi A; Stinson, Jeffrey R; Zhang, Yuan; Abbott, Jordan K; Weinreich, Michael A; Hauk, Pia J; Reynolds, Paul R; Lyons, Jonathan J; Nelson, Celeste G; Ruffo, Elisa; Dorjbal, Batsukh; Glauzy, Salomé; Yamakawa, Natsuko; Arjunaraja, Swadhinya; Voss, Kelsey; Stoddard, Jennifer; Niemela, Julie; Zhang, Yu; Rosenzweig, Sergio D; McElwee, Joshua J; DiMaggio, Thomas; Matthews, Helen F; Jones, Nina; Stone, Kelly D; Palma, Alejandro; Oleastro, Matías; Prieto, Emma; Bernasconi, Andrea R; Dubra, Geronimo; Danielian, Silvia; Zaiat, Jonathan; Marti, Marcelo A; Kim, Brian; Cooper, Megan A; Romberg, Neil D; Meffre, Eric; Gelfand, Erwin W; Snow, Andrew L; Milner, Joshua D

2017-01-01

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Via next generation sequencing on a cohort of patients with severe atopic dermatitis, some with comorbid infections, we found 8 individuals from 4 families with novel heterozygous mutations in CARD11, a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant expression constructs into T cell lines demonstrated both loss of function and dominant interfering activity upon antigen receptor-induced NF-κB and mTORC1 activation. Patient T-cells had similar defects, as well as diminished IFN-γ cytokine production. The mTORC1 and IFN-γ production defects could be partially rescued by supplementing with glutamine, which requires CARD11 for import into T cells. Our findings indicate a single hypomorphic gene mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis. PMID:28628108

Calpain 12 Function Revealed through the Study of an Atypical Case of Autosomal Recessive Congenital Ichthyosis.

PubMed

Bochner, Ron; Samuelov, Liat; Sarig, Ofer; Li, Qiaoli; Adase, Christopher A; Isakov, Ofer; Malchin, Natalia; Vodo, Dan; Shayevitch, Ronna; Peled, Alon; Yu, Benjamin D; Fainberg, Gilad; Warshauer, Emily; Adir, Noam; Erez, Noam; Gat, Andrea; Gottlieb, Yehonatan; Rogers, Tova; Pavlovsky, Mor; Goldberg, Ilan; Shomron, Noam; Sandilands, Aileen; Campbell, Linda E; MacCallum, Stephanie; McLean, W H Irwin; Ast, Gil; Gallo, Richard L; Uitto, Jouni; Sprecher, Eli

2017-02-01

Congenital erythroderma is a rare and often life-threatening condition, which has been shown to result from mutations in several genes encoding important components of the epidermal differentiation program. Using whole exome sequencing, we identified in a child with congenital exfoliative erythroderma, hypotrichosis, severe nail dystrophy and failure to thrive, two heterozygous mutations in ABCA12 (c.2956C>T, p.R986W; c.5778+2T>C, p. G1900Mfs*16), a gene known to be associated with two forms of ichthyosis, autosomal recessive congenital ichthyosis, and harlequin ichthyosis. Because the patient displayed an atypical phenotype, including severe hair and nail manifestations, we scrutinized the exome sequencing data for additional potentially deleterious genetic variations in genes of relevance to the cornification process. Two mutations were identified in CAPN12, encoding a member of the calpain proteases: a paternal missense mutation (c.1511C>A; p.P504Q) and a maternal deletion due to activation of a cryptic splice site in exon 9 of the gene (c.1090_1129del; p.Val364Lysfs*11). The calpain 12 protein was found to be expressed in both the epidermis and hair follicle of normal skin, but its expression was dramatically reduced in the patient's skin. The downregulation of capn12 expression in zebrafish was associated with abnormal epidermal morphogenesis. Small interfering RNA knockdown of CAPN12 in three-dimensional human skin models was associated with acanthosis, disorganized epidermal architecture, and downregulation of several differentiation markers, including filaggrin. Accordingly, filaggrin expression was almost absent in the patient skin. Using ex vivo live imaging, small interfering RNA knockdown of calpain 12 in skin from K14-H2B GFP mice led to significant hair follicle catagen transformation compared with controls. In summary, our results indicate that calpain 12 plays an essential role during epidermal ontogenesis and normal hair follicle cycling and that its absence may aggravate the clinical manifestations of ABCA12 mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

PubMed Central

Genschel, Jochen; Kadyrova, Lyudmila Y.; Iyer, Ravi R.; Dahal, Basanta K.; Kadyrov, Farid A.; Modrich, Paul

2017-01-01

Eukaryotic MutLα (mammalian MLH1–PMS2 heterodimer; MLH1–PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif (723QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif (723AKLIIP) with an exo1Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell. PMID:28439008

Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair.

PubMed

Genschel, Jochen; Kadyrova, Lyudmila Y; Iyer, Ravi R; Dahal, Basanta K; Kadyrov, Farid A; Modrich, Paul

2017-05-09

Eukaryotic MutLα (mammalian MLH1-PMS2 heterodimer; MLH1-PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721 QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif ( 723 QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif ( 723 AKLIIP) with an exo1 Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell.

Activating PIK3CA mutations coexist with BRAF or NRAS mutations in a limited fraction of melanomas.

PubMed

Manca, Antonella; Lissia, Amelia; Capone, Mariaelena; Ascierto, Paolo A; Botti, Gerardo; Caracò, Corrado; Stanganelli, Ignazio; Colombino, Maria; Sini, MariaCristina; Cossu, Antonio; Palmieri, Giuseppe

2015-01-28

Activated PI3K-AKT pathway may contribute to decrease sensitivity to inhibitors of key pathogenetic effectors (mutated BRAF, active NRAS or MEK) in melanoma. Functional alterations are deeply involved in PI3K-AKT activation, with a minimal role reported for mutations in PIK3CA, the catalytic subunit of the PI3K gene. We here assessed the prevalence of the coexistence of BRAF/NRAS and PIK3CA mutations in a series of melanoma samples. A total of 245 tumor specimens (212 primary melanomas and 33 melanoma cell lines) was screened for mutations in BRAF, NRAS, and PIK3CA genes by automated direct sequencing. Overall, 110 (44.9%) samples carried mutations in BRAF, 26 (10.6%) in NRAS, and 24 (9.8%) in PIK3CA. All identified PIK3CA mutations have been reported to induce PI3K activation; those detected in cultured melanomas were investigated for their interference with the antiproliferative activity of the BRAF-mutant inhibitor vemurafenib. A reduced suppression in cell growth was observed in treated cells carrying both BRAF and PIK3CA mutations as compared with those presenting a mutated BRAF only. Among the analysed melanomas, 12/245 (4.9%) samples presented the coexistence of PIK3CA and BRAF/NRAS mutations. Our study further suggests that PIK3CA mutations account for a small fraction of PI3K pathway activation and have a limited impact in interfering with the BRAF/NRAS-driven growth in melanoma.

EGFR, KRAS, and BRAF mutational profiles of female patients with micropapillary predominant invasive lung adenocarcinoma

PubMed

Demirağ, Funda; Yılmaz, Aydın; Yılmaz Demirci, Nilgün; Yılmaz, Ülkü; Erdoğan, Yurdanur

2017-11-13

Background/aim: This study aimed to analyze EGFR, KRAS, and BRAF mutations in females with micropapillary predominant invasive lung adenocarcinoma and their relationships with immunohistochemical and clinicopathological patterns.Materials and methods: A total of 15 females with micropapillary lung adenocarcinoma were selected. Mutational analysis of the EGFR, KRAS, and BRAF genes was carried out. Information regarding the demographic data, tumor size, treatment, and survival time for each patient was collated, and the predominant cell type, secondary architectural growth patterns, psammoma bodies, necrosis, and visceral pleural and angiolymphatic invasions were evaluated.Results: We identified EGFR mutation in six cases, KRAS mutation in three cases, and BRAF mutation in one case. EGFR, c-kit, VEGFR, and bcl-2 positivity was observed in ten, seven, four, and six cases, respectively. All cases were positive for VEGF (strong positivity in 11 cases and weak positivity in four cases) and bcl-2 (strong positivity in nine cases and weak positivity in six cases). Seven (46.6%) cases were positive for c-kit and 10 (66.6%) cases were positive for EGFR. Conclusion: EGFR mutation occurred at a higher incidence rate in micropapillary predominant invasive adenocarcinoma than has previously been found in conventional lung adenocarcinomas. KRAS mutation was observed as having a similar frequency to what was previously observed, but the frequency of BRAF mutation was lower than previously reported.

Exploring environmental causes of altered ras effects: fragmentation plus integration?

PubMed

Porta, Miquel; Ayude, Daniel; Alguacil, Juan; Jariod, Manuel

2003-02-01

Mutations in ras genes are the most common abnormality of oncogenes in human cancer and a major example of activation by point mutation. Experimental and epidemiological studies support the notion that Ki-ras activation and expression may be chemically related. We discuss the potential role of several environmental compounds in the induction or promotion of ras mutations in humans, with a focus on exocrine pancreatic cancer, the human tumor with the highest prevalence at diagnosis of Ki-ras mutations. Organochlorine compounds, organic solvents, and coffee compounds may play an indirect role in causing Ki-ras mutations, rather than as direct inducers of the mutations. Although for some organochlorine compounds the induction of point mutations in ras oncogenes cannot be excluded, it seems more likely that the effects of these compounds are mediated through nongenomic or indirectly genotoxic mechanisms of action. Organic solvents also may act via enzymatic induction of ras mutagens or by providing a proliferation advantage to ras-mutated cell clones. In exocrine pancreatic cancer, caffeine, other coffee compounds, or other factors with which coffee drinking is associated could modulate Ki-ras activation by interfering with DNA repair, cell-cycle checkpoints, and apoptosis. Asbestos, cigarette smoking, and some dietary factors also may be involved in the initiation or the promotion of Ki-ras mutations in lung and colon cancers. Further development of the mechanistic scenarios proposed here could contribute to a meaningful integration of biological, clinical, and environmental knowledge on the causes of altered ras effects. Copyright 2003 Wiley-Liss, Inc.

Minimum Variance Distortionless Response Beamformer with Enhanced Nulling Level Control via Dynamic Mutated Artificial Immune System

PubMed Central

Kiong, Tiong Sieh; Salem, S. Balasem; Paw, Johnny Koh Siaw; Sankar, K. Prajindra

2014-01-01

In smart antenna applications, the adaptive beamforming technique is used to cancel interfering signals (placing nulls) and produce or steer a strong beam toward the target signal according to the calculated weight vectors. Minimum variance distortionless response (MVDR) beamforming is capable of determining the weight vectors for beam steering; however, its nulling level on the interference sources remains unsatisfactory. Beamforming can be considered as an optimization problem, such that optimal weight vector should be obtained through computation. Hence, in this paper, a new dynamic mutated artificial immune system (DM-AIS) is proposed to enhance MVDR beamforming for controlling the null steering of interference and increase the signal to interference noise ratio (SINR) for wanted signals. PMID:25003136

Minimum variance distortionless response beamformer with enhanced nulling level control via dynamic mutated artificial immune system.

PubMed

Kiong, Tiong Sieh; Salem, S Balasem; Paw, Johnny Koh Siaw; Sankar, K Prajindra; Darzi, Soodabeh

2014-01-01

In smart antenna applications, the adaptive beamforming technique is used to cancel interfering signals (placing nulls) and produce or steer a strong beam toward the target signal according to the calculated weight vectors. Minimum variance distortionless response (MVDR) beamforming is capable of determining the weight vectors for beam steering; however, its nulling level on the interference sources remains unsatisfactory. Beamforming can be considered as an optimization problem, such that optimal weight vector should be obtained through computation. Hence, in this paper, a new dynamic mutated artificial immune system (DM-AIS) is proposed to enhance MVDR beamforming for controlling the null steering of interference and increase the signal to interference noise ratio (SINR) for wanted signals.

Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

PubMed Central

Mao, Cheng-Qiong; Xiong, Meng-Hua; Liu, Yang; Shen, Song; Du, Xiao-Jiao; Yang, Xian-Zhu; Dou, Shuang; Zhang, Pei-Zhuo; Wang, Jun

2014-01-01

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. PMID:24496383

Congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations.

PubMed

Guergueltcheva, Velina; Müller, Juliane S; Dusl, Marina; Senderek, Jan; Oldfors, Anders; Lindbergh, Christopher; Maxwell, Susan; Colomer, Jaume; Mallebrera, Cecilia Jimenez; Nascimento, Andres; Vilchez, Juan J; Muelas, Nuria; Kirschner, Janbernd; Nafissi, Shahriar; Kariminejad, Ariana; Nilipour, Yalda; Bozorgmehr, Bita; Najmabadi, Hossein; Rodolico, Carmelo; Sieb, Jörn P; Schlotter, Beate; Schoser, Benedikt; Herrmann, Ralf; Voit, Thomas; Steinlein, Ortrud K; Najafi, Abdolhamid; Urtizberea, Andoni; Soler, Doriette M; Muntoni, Francesco; Hanna, Michael G; Chaouch, Amina; Straub, Volker; Bushby, Kate; Palace, Jacqueline; Beeson, David; Abicht, Angela; Lochmüller, Hanns

2012-05-01

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders of the neuromuscular junction. A difficult to diagnose subgroup of CMS is characterised by proximal muscle weakness and fatigue while ocular and facial involvement is only minimal. DOK7 mutations have been identified as causing the disorder in about half of the cases. More recently, using classical positional cloning, we have identified mutations in a previously unrecognised CMS gene, GFPT1, in a series of DOK7-negative cases. However, detailed description of clinical features of GFPT1 patients has not been reported yet. Here we describe the clinical picture of 24 limb-girdle CMS (LG-CMS) patients and pathological findings of 18 of them, all carrying GFPT1 mutations. Additional patients with CMS, but without tubular aggregates, and patients with non-fatigable weakness with tubular aggregates were also screened. In most patients with GFPT1 mutations, onset of the disease occurs in the first decade of life with characteristic limb-girdle weakness and fatigue. A common feature was beneficial and sustained response to acetylcholinesterase inhibitor treatment. Most of the patients who had a muscle biopsy showed tubular aggregates in myofibers. Analysis of endplate morphology in one of the patients revealed unspecific abnormalities. Our study delineates the phenotype of CMS associated with GFPT1 mutations and expands the understanding of neuromuscular junction disorders. As tubular aggregates in context of a neuromuscular transmission defect appear to be highly indicative, we suggest calling this condition congenital myasthenic syndrome with tubular aggregates (CMS-TA).

Therapeutic targeting of RNA splicing in myelodysplasia.

PubMed

Kim, Young Joon; Abdel-Wahab, Omar

2017-07-01

Genomic analysis of patients with myelodysplastic syndromes (MDS) has identified that mutations within genes encoding RNA splicing factors represent the most common class of genetic alterations in MDS. These mutations primarily affect SF3B1, SRSF2, U2AF1, and ZRSR2. Current data suggest that these mutations perturb RNA splicing catalysis in a manner distinct from loss of function but how exactly the global changes in RNA splicing imparted by these mutations result in MDS is not well delineated. At the same time, cells bearing mutations in RNA splicing factors are exquisitely dependent on the presence of the remaining wild-type (WT) allele to maintain residual normal splicing for cell survival. The high frequency of these mutations in MDS, combined with their mutual exclusivity and noteworthy dependence on the WT allele, make targeting RNA splicing attractive in MDS. To this end, two promising therapeutic approaches targeting RNA splicing are being tested clinically currently. These include molecules targeting core RNA splicing catalysis by interfering with the ability of the SF3b complex to interact with RNA, as well as molecules degrading the auxiliary RNA splicing factor RBM39. The preclinical and clinical evaluation of these compounds are discussed here in addition to their potential as therapies for spliceosomal mutant MDS. Copyright © 2017 Elsevier Inc. All rights reserved.

Complex pattern of immune evasion in MSI colorectal cancer.

PubMed

Ozcan, Mine; Janikovits, Jonas; von Knebel Doeberitz, Magnus; Kloor, Matthias

2018-01-01

Mismatch repair (MMR)-deficient cancers accumulate multiple insertion/deletion mutations at coding microsatellites (cMS), which give rise to frameshift peptide neoantigens. The high mutational neoantigen load of MMR-deficient cancers is reflected by pronounced anti-tumoral immune responses of the host and high responsiveness towards immune checkpoint blockade. However, immune evasion mechanisms can interfere with the immune response against MMR-deficient tumors. We here performed a comprehensive analysis of immune evasion in MMR-deficient colorectal cancers, focusing on HLA class I-mediated antigen presentation. 72% of MMR-deficient colorectal cancers of the DFCI database harbored alterations affecting genes involved in HLA class I-mediated antigen presentation, and 54% of these mutations were predicted to abrogate function. Mutations affecting the HLA class I transactivator NLRC5 were observed as a potential new immune evasion mechanism in 26% (6% abrogating) of the analyzed tumors. NLRC5 mutations in MMR-deficient cancers were associated with decreased levels of HLA class I antigen expression. In summary, the majority of MMR-deficient cancers display mutations interfering with HLA class I antigen presentation that reflect active immune surveillance and immunoselection during tumor development. Clinical studies focusing on immune checkpoint blockade in MSI cancer should account for the broad variety of immune evasion mechanisms as potential biomarkers of therapy success.

Adaptive Array for Weak Interfering Signals: Geostationary Satellite Experiments. M.S. Thesis

NASA Technical Reports Server (NTRS)

Steadman, Karl

1989-01-01

The performance of an experimental adaptive array is evaluated using signals from an existing geostationary satellite interference environment. To do this, an earth station antenna was built to receive signals from various geostationary satellites. In these experiments the received signals have a frequency of approximately 4 GHz (C-band) and have a bandwidth of over 35 MHz. These signals are downconverted to a 69 MHz intermediate frequency in the experimental system. Using the downconverted signals, the performance of the experimental system for various signal scenarios is evaluated. In this situation, due to the inherent thermal noise, qualitative instead of quantitative test results are presented. It is shown that the experimental system can null up to two interfering signals well below the noise level. However, to avoid the cancellation of the desired signal, the use a steering vector is needed. Various methods to obtain an estimate of the steering vector are proposed.

An elderly-onset limb girdle muscular dystrophy type 1B (LGMD1B) with pseudo-hypertrophy of paraspinal muscles.

PubMed

Furuta, Mitsuru; Sumi-Akamaru, Hisae; Takahashi, Masanori P; Hayashi, Yukiko K; Nishino, Ichizo; Mochizuki, Hideki

2016-09-01

Mutations in LMNA, encoding A-type lamins, lead to diverse disorders, collectively called "laminopathies," which affect the striated muscle, cardiac muscle, adipose tissue, skin, peripheral nerve, and premature aging. We describe a patient with limb-girdle muscular dystrophy type 1B (LGMD1B) carrying a heterozygous p.Arg377His mutation in LMNA, in whom skeletal muscle symptom onset was at the age of 65 years. Her weakness started at the erector spinae muscles, which showed marked pseudo-hypertrophy even at the age of 72 years. Her first episode of syncope was at 44 years; however, aberrant cardiac conduction was not revealed until 60 years. The p.Arg377His mutation has been previously reported in several familial LMNA-associated myopathies, most of which showed muscle weakness before the 6th decade. This is the first report of pseudo-hypertrophy of paravertebral muscles in LMNA-associated myopathies. The pseudo-hypertrophy of paravertebral muscles and the elderly-onset of muscle weakness make this case unique and reportable. Copyright © 2016 Elsevier B.V. All rights reserved.

Adult onset Leigh syndrome with mitochondrial DNA 8344 A>G mutation.

PubMed

Han, Jee-Young; Sung, Jung-Joon; Park, Hong-Kyun; Yoon, Byung-Nam; Lee, Kwang-Woo

2014-11-01

We report a pedigree of adult-onset Leigh syndrome (LS) with mitochondrial mutation 8344 A>G. A 38-year-old woman presented with optic neuropathy, weakness and cognitive impairment. Family history of optic neuropathy and systemic involvement was suggestive of mitochondrial encephalopathy. Genetic and radiologic studies showed m.8344 A>G mutation with characteristics of LS. To our knowledge this is the first case of adult-onset LS demonstrating the m.8344 A>G mutation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Preliminary Evaluation of the Effect of Investigational Ebola Virus Disease Treatments on Viral Genome Sequences.

PubMed

Whitmer, Shannon L M; Albariño, César; Shepard, Samuel S; Dudas, Gytis; Sheth, Mili; Brown, Shelley C; Cannon, Deborah; Erickson, Bobbie R; Gibbons, Aridth; Schuh, Amy; Sealy, Tara; Ervin, Elizabeth; Frace, Mike; Uyeki, Timothy M; Nichol, Stuart T; Ströher, Ute

2016-10-15

 Several patients with Ebola virus disease (EVD) managed in the United States have received ZMapp monoclonal antibodies, TKM-Ebola small interfering RNA, brincidofovir, and/or convalescent plasma as investigational therapeutics.  To investigate whether treatment selected for Ebola virus (EBOV) mutations conferring resistance, viral sequencing was performed on RNA extracted from clinical blood specimens from patients with EVD following treatment, and putative viral targets were analyzed.  We observed no major or minor EBOV mutations within regions targeted by therapeutics.  This small subset of patients and clinical specimens suggests that evolution of resistance is not a direct consequence of antiviral treatment. As EVD antiviral treatments are introduced into wider use, it is essential that continuous viral full-genome surveillance is performed, to monitor for the emergence of escape mutations. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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

Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas themore » COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.« less

In-silico analysis for RNA-interference mechanism of α-synuclein to treat Parkinson's disease.

PubMed

Seema, S; Seenivasagam, R; Hemavathi, K

2013-01-01

Parkinson's Disease (PD) causing mutations in α-synuclein gene are ALA30PRO, GLU46LYS and ALA53THR. The conformational changes in proteins with respect to all the three mutations were analysed. These were used to predict the structures of Short Interfering RNA (siRNA) antisense strand and siRNA region. The siRNA binds with the argonaute protein forming RNA Induced Silencing Complex (RISC). Then, siRNA antisense-strand was attached to RISC. The structure of dicer (RNase-III-enzyme) cleaves double-stranded RNA (dsRNA) into two siRNA-strands. Incorporation of single siRNA-strand into RISC guides to pair with the complementary α-synuclein target-messenger RNA (mRNA) thereby enabling it to cleave the target.

Genetic and mechanistic evaluation for the weak A phenotype in Ael blood type with IVS6 + 5G>A ABO gene mutation.

PubMed

Chen, D-P; Sun, C-F; Ning, H-C; Peng, C-T; Wang, W-T; Tseng, C-P

2015-01-01

Ael is a rare blood type that is characterized by weak agglutination of RBCs when reacts with anti-A antibody in adsorption-elution test. Although IVS6 + 5G→A mutation is known to associate with the Ael blood type, genetic and mechanistic evaluation for the weak agglutination of Ael with IVS6 + 5G→A mutation has not yet been completely addressed. In this study, five cases of confirmed Ael individuals were analysed. The cDNAs for the A(el) alleles were obtained by cloning method for sequence analyses. The erythroleukemia K562 cells were used as the cell study model and were transfected with the A(el) expression construct. Flow cytometry analysis was then performed to determine the levels of surface antigen expression. The results indicated that IVS6 + 5G→A attributes to all cases of Ael . RT-PCR analyses revealed the presence of at least 10 types of aberrant A(el) splicing transcripts. Most of the transcripts caused early termination and produced non-functional protein during translation. Nevertheless, the transcript without exons 5-6 was predicted to generate functional Ael glycosyltransferase lacking 57 amino acids at the N-terminal segment. When the exons 5-6 deletion transcript was stably expressed in the K562 cells, weak agglutination of the cells can be induced by adding anti-A antibody followed by adsorption-elution test. This study demonstrates that aberrant splicing of A transcripts contributes to weak A expression and the weak agglutination of Ael -RBCs, adding to the complexity for the regulatory mechanisms of ABO gene expression. © 2014 International Society of Blood Transfusion.

An R132H Mutation in Isocitrate Dehydrogenase 1 Enhances p21 Expression and Inhibits Phosphorylation of Retinoblastoma Protein in Glioma Cells

PubMed Central

Miyata, Satsuki; Urabe, Masashi; Gomi, Akira; Nagai, Mutsumi; Yamaguchi, Takashi; Tsukahara, Tomonori; Mizukami, Hiroaki; Kume, Akihiro; Ozawa, Keiya; Watanabe, Eiju

2013-01-01

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1R132H-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1R132H-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progressionof the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation. PMID:24077277

An R132H mutation in isocitrate dehydrogenase 1 enhances p21 expression and inhibits phosphorylation of retinoblastoma protein in glioma cells.

PubMed

Miyata, Satsuki; Urabe, Masashi; Gomi, Akira; Nagai, Mutsumi; Yamaguchi, Takashi; Tsukahara, Tomonori; Mizukami, Hiroaki; Kume, Akihiro; Ozawa, Keiya; Watanabe, Eiju

2013-01-01

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1(R132H)-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1(R132H)-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progression of the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation.

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

PubMed Central

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

2016-01-01

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

Recessive and dominant mutations in COL12A1 cause a novel EDS/myopathy overlap syndrome in humans and mice

PubMed Central

Zou, Yaqun; Zwolanek, Daniela; Izu, Yayoi; Gandhy, Shreya; Schreiber, Gudrun; Brockmann, Knut; Devoto, Marcella; Tian, Zuozhen; Hu, Ying; Veit, Guido; Meier, Markus; Stetefeld, Jörg; Hicks, Debbie; Straub, Volker; Voermans, Nicol C.; Birk, David E.; Barton, Elisabeth R.; Koch, Manuel; Bönnemann, Carsten G.

2014-01-01

Collagen VI-related myopathies are disorders of connective tissue presenting with an overlap phenotype combining clinical involvement from the muscle and from the connective tissue. Not all patients displaying related overlap phenotypes between muscle and connective tissue have mutations in collagen VI. Here, we report a homozygous recessive loss of function mutation and a de novo dominant mutation in collagen XII (COL12A1) as underlying a novel overlap syndrome involving muscle and connective tissue. Two siblings homozygous for a loss of function mutation showed widespread joint hyperlaxity combined with weakness precluding independent ambulation, while the patient with the de novo missense mutation was more mildly affected, showing improvement including the acquisition of walking. A mouse model with inactivation of the Col12a1 gene showed decreased grip strength, a delay in fiber-type transition and a deficiency in passive force generation while the muscle seems more resistant to eccentric contraction induced force drop, indicating a role for a matrix-based passive force-transducing elastic element in the generation of the weakness. This new muscle connective tissue overlap syndrome expands on the emerging importance of the muscle extracellular matrix in the pathogenesis of muscle disease. PMID:24334604

Evaluation of IDH1 status in diffusely infiltrating gliomas by immunohistochemistry using anti-mutant and wild type IDH1 antibodies.

PubMed

Ikota, Hayato; Nobusawa, Sumihito; Arai, Hideo; Kato, Yukinari; Ishizawa, Keisuke; Hirose, Takanori; Yokoo, Hideaki

2015-10-01

Glioma cells with the isocitrate dehydrogenase (IDH) 1 G395A mutation are strongly immunopositive for mIDH1(R132H), an antibody against mutant IDH1(R132H) (clone H09). However, we encountered some gliomas which were ambiguously positive for mIDH1(R132H) despite having the IDH1 G395A mutation. The aim of this study was to establish an evaluation procedure of IDH1 status by immunohistochemistry. Forty-three diffusely infiltrating gliomas were studied, and four of eight anaplastic oligoastrocytomas with the IDH1 G395A mutation were modestly or weakly positive for both the mIDH1(R132H) and an antibody against wild type IDH1, RcMab-1. Based on our staining results, the IDH1 expression of both wild and mutated types seemed to be codominant and also to be evenly suppressed under a certain condition. We propose a procedure for determining IDH1 status. If a glioma is weakly positive for mIDH1(R132H), immunohistochemistry for RcMab-1 should be performed. If the tumor cells are strongly positive for RcMab-1, the IDH1 G395A mutation is judged to be absent on the grounds that IDH1 expression is not suppressed. If the tumor cells are weakly positive for both mIDH1(R132H) and RcMab-1, then a conclusion should be made after DNA sequencing. This procedure is useful for practical evaluation of IDH1 status.

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

NASA Astrophysics Data System (ADS)

Berntsen, K. Nikolaj

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

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

PubMed Central

Goldgof, Gregory M.; Durrant, Jacob D.; Ottilie, Sabine; Vigil, Edgar; Allen, Kenneth E.; Gunawan, Felicia; Kostylev, Maxim; Henderson, Kiersten A.; Yang, Jennifer; Schenken, Jake; LaMonte, Gregory M.; Manary, Micah J.; Murao, Ayako; Nachon, Marie; Stanhope, Rebecca; Prescott, Maximo; McNamara, Case W.; Slayman, Carolyn W.; Amaro, Rommie E.; Suzuki, Yo; Winzeler, Elizabeth A.

2016-01-01

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity. PMID:27291296

A novel mutation of the adrenocorticotropin receptor (ACTH-R) gene in a family with the syndrome of isolated glucocorticoid deficiency, but no ACTH-R abnormalities in two families with the triple A syndrome

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

Tsigos, C.; Arai, K.; Latronico, A.C.

1995-07-01

Isolated glucocorticoid deficiency (IGD) is an autosomal recessive disorder characterized by primary adrenocortical insufficiency, usually without mineralocorticoid deficiency. Occasionally, the disorder is associated with alacrima and achalasia of the esophagus (triple A syndrome), suggesting potential heterogeneity in its etiology. Mutations in the ACTH receptor gene have been reported in several families with IGD. We have amplified and directly sequenced the entire intronless ACTH receptor gene in 1 other family with IGD and 2 famlies with triple A syndrome. The proband with IGD was a homozygote for an A {r_arrow}G substitution, changing tyrosine 254 to cysteine in the third extracellular loopmore » of the receptor protein, probably interfering with ligand binding. Both of her parents were heterozygotes for this mutation, which was not detected in 100 normal alleles. No mutations were identified in the entire coding area of the ACTH receptor in the 2 families with triple A syndrome, supporting the idea of a developmental or postreceptor defect in this syndrome. 19 refs., 1 fig.« less

The MicroRNA390/TRANS-ACTING SHORT INTERFERING RNA3 Module Mediates Lateral Root Growth under Salt Stress via the Auxin Pathway.

PubMed

He, Fu; Xu, Changzheng; Fu, Xiaokang; Shen, Yun; Guo, Li; Leng, Mi; Luo, Keming

2018-06-01

Salt-induced developmental plasticity in a plant root system strongly depends on auxin signaling. However, the molecular events underlying this process are poorly understood. MicroRNA390 ( miR390 ), trans-actin small interfering RNA s ( tasiRNA s), and AUXIN RESPONSE FACTORs ( ARFs ) form a regulatory module involved in controlling lateral root (LR) growth. Here, we found that miR390 expression was strongly induced by exposure to salt during LR formation in poplar ( Populus spp.) plants. miR390 overexpression stimulated LR development and increased salt tolerance, whereas miR390 knockdown caused by a short tandem target mimic repressed LR growth and compromised salt resistance. ARF3.1 , ARF3.2 , and ARF4 expression was inhibited significantly by the presence of salt, and transcript abundance was decreased dramatically in the miR390 -overexpressing line but increased in the miR390 -knockdown line. Constitutive expression of ARF4m harboring mutated trans-acting small interfering ARF -binding sites removed the salt resistance of the miR390 overexpressors. miR390 positively regulated auxin signaling in LRs subjected to salt, but ARF4 inhibited auxin signaling. Salinity stabilized the poplar Aux/IAA repressor INDOLE-3-ACETIC ACID17.1, and overexpression of an auxin/salt-resistant form of this repressor suppressed LR growth in miR390 -overexpressing and ARF4 -RNA interfering lines in the presence of salt. Thus, the miR390/TAS3/ARFs module is a key regulator, via modulating the auxin pathway, of LR growth in poplar subjected to salt stress. © 2018 American Society of Plant Biologists. All rights reserved.

Conflicting Selection Pressures Will Constrain Viral Escape from Interfering Particles: Principles for Designing Resistance-Proof Antivirals.

PubMed

Rast, Luke I; Rouzine, Igor M; Rozhnova, Ganna; Bishop, Lisa; Weinberger, Ariel D; Weinberger, Leor S

2016-05-01

The rapid evolution of RNA-encoded viruses such as HIV presents a major barrier to infectious disease control using conventional pharmaceuticals and vaccines. Previously, it was proposed that defective interfering particles could be developed to indefinitely control the HIV/AIDS pandemic; in individual patients, these engineered molecular parasites were further predicted to be refractory to HIV's mutational escape (i.e., be 'resistance-proof'). However, an outstanding question has been whether these engineered interfering particles-termed Therapeutic Interfering Particles (TIPs)-would remain resistance-proof at the population-scale, where TIP-resistant HIV mutants may transmit more efficiently by reaching higher viral loads in the TIP-treated subpopulation. Here, we develop a multi-scale model to test whether TIPs will maintain indefinite control of HIV at the population-scale, as HIV ('unilaterally') evolves toward TIP resistance by limiting the production of viral proteins available for TIPs to parasitize. Model results capture the existence of two intrinsic evolutionary tradeoffs that collectively prevent the spread of TIP-resistant HIV mutants in a population. First, despite their increased transmission rates in TIP-treated sub-populations, unilateral TIP-resistant mutants are shown to have reduced transmission rates in TIP-untreated sub-populations. Second, these TIP-resistant mutants are shown to have reduced growth rates (i.e., replicative fitness) in both TIP-treated and TIP-untreated individuals. As a result of these tradeoffs, the model finds that TIP-susceptible HIV strains continually outcompete TIP-resistant HIV mutants at both patient and population scales when TIPs are engineered to express >3-fold more genomic RNA than HIV expresses. Thus, the results provide design constraints for engineering population-scale therapies that may be refractory to the acquisition of antiviral resistance.

Progesterone receptor membrane component-1 (PGRMC1) is the mediator of progesterone's antiapoptotic action in spontaneously immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic acid treatment and functional analysis of PGRMC1 mutations.

PubMed

Peluso, John J; Romak, Jonathan; Liu, Xiufang

2008-02-01

Progesterone (P4) receptor membrane component-1 (PGRMC1) and its binding partner, plasminogen activator inhibitor 1 RNA binding protein (PAIRBP1) are thought to form a complex that functions as membrane receptor for P4. The present investigations confirm PGRMC1's role in this membrane receptor complex by demonstrating that depleting PGMRC1 with PGRMC1 small interfering RNA results in a 60% decline in [(3)H]P4 binding and the loss of P4's antiapoptotic action. Studies conducted on partially purified GFP-PGRMC1 fusion protein indicate that [(3)H]P4 specifically binds to PGRMC1 at a single site with an apparent K(d) of about 35 nm. In addition, experiments using various deletion mutations reveal that the entire PGRMC1 molecule is required for maximal [(3)H]P4 binding and P4 responsiveness. Analysis of the binding data also suggests that the P4 binding site is within a segment of PGRMC1 that is composed of the transmembrane domain and the initial segment of the C terminus. Interestingly, PAIRBP1 appears to bind to the C terminus between amino acids 70-130, which is distal to the putative P4 binding site. Taken together, these data provide compelling evidence that PGRMC1 is the P4 binding protein that mediates P4's antiapoptotic action. Moreover, the deletion mutation studies indicate that each domain of PGRMC1 plays an essential role in modulating PGRMC1's capacity to both bind and respond to P4. Additional studies are required to more precisely delineate the role of each PGRMC1 domain in transducing P4's antiapoptotic action.

The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome.

PubMed

Cossins, Judith; Liu, Wei Wei; Belaya, Katsiaryna; Maxwell, Susan; Oldridge, Michael; Lester, Tracy; Robb, Stephanie; Beeson, David

2012-09-01

Congenital myasthenic syndromes (CMS) are a group of inherited diseases that affect synaptic transmission at the neuromuscular junction and result in fatiguable muscle weakness. A subgroup of CMS patients have a recessively inherited limb-girdle pattern of weakness caused by mutations in DOK7. DOK7 encodes DOK7, an adaptor protein that is expressed in the skeletal muscle and heart and that is essential for the development and maintenance of the neuromuscular junction. We have screened the DOK7 gene for mutations by polymerase chain reaction amplification and bi-directional sequencing of exonic and promoter regions and performed acetylcholine receptor (AChR) clustering assays and used exon trapping to determine the pathogenicity of detected variants. Approximately 18% of genetically diagnosed CMSs in the UK have mutations in DOK7, with mutations in this gene identified in more than 60 kinships to date. Thirty-four different pathogenic mutations were identified as well as 27 variants likely to be non-pathogenic. An exon 7 frameshift duplication c.1124_1127dupTGCC is commonly found in at least one allele. We analyse the effect of the common frameshift c.1124_1127dupTGCC and show that 10/11 suspected missense mutations have a deleterious effect on AChR clustering. We identify for the first time homozygous or compound heterozygous mutations that are localized 5' to exon 7. In addition, three silent variants in the N-terminal half of DOK7 are predicted to alter the splicing of the DOK7 RNA transcript. The DOK7 gene is highly polymorphic, and within these many variants, we define a spectrum of mutations that can underlie DOK7 CMS that will inform in managing this disorder.

Mental retardation linked to mutations in the HSD17B10 gene interfering with neurosteroid and isoleucine metabolism

PubMed Central

Yang, Song-Yu; He, Xue-Ying; Olpin, Simon E.; Sutton, Vernon R.; McMenamin, Joe; Philipp, Manfred; Denman, Robert B.; Malik, Mazhar

2009-01-01

Mutations in the HSD17B10 gene were identified in two previously described mentally retarded males. A point mutation c.776G>C was found from a survivor (SV), whereas a potent mutation, c.419C>T, was identified in another deceased case (SF) with undetectable hydroxysteroid (17β) dehydrogenase 10 (HSD10) activity. Protein levels of mutant HSD10(R130C) in patient SF and HSD10(E249Q) in patient SV were about half that of HSD10 in normal controls. The E249Q mutation appears to affect HSD10 subunit interactions, resulting in an allosteric regulatory enzyme. For catalyzing the oxidation of allopregnanolone by NAD+ the Hill coefficient of the mutant enzyme is ≈1.3. HSD10(E249Q) was unable to catalyze the dehydrogenation of 2-methyl-3-hydroxybutyryl-CoA and the oxidation of allopregnanolone, a positive modulator of the γ-aminobutyric acid type A receptor, at low substrate concentrations. Neurosteroid homeostasis is critical for normal cognitive development, and there is increasing evidence that a blockade of isoleucine catabolism alone does not commonly cause developmental disabilities. The results support the theory that an imbalance in neurosteroid metabolism could be a major cause of the neurological handicap associated with hydroxysteroid (17β) dehydrogenase 10 deficiency. PMID:19706438

A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation.

PubMed

Bleichert, Franziska; Balasov, Maxim; Chesnokov, Igor; Nogales, Eva; Botchan, Michael R; Berger, James M

2013-10-08

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.

Cellstat--A continuous culture system of a bacteriophage for the study of the mutation rate and the selection process at the DNA level

NASA Astrophysics Data System (ADS)

Husimi, Yuzuru; Nishigaki, Koichi; Kinoshita, Yasunori; Tanaka, Toyosuke

1982-04-01

A bacteriophage is continuously cultured in the flow of the host bacterial cell under the control of a minicomputer. In the culture, the population of the noninfected cell is kept constant by the endogeneous regulation mechanism, so it is called the ''cellstat'' culture. Due to the high dilution rate of the host cell, the mutant cell cannot be selected in the cellstat. Therefore, the cellstat is suitable for the study of the mutation rate and the selection process of a bacteriophage under well-defined environmental conditions (including physiological condition of the host cell) without being interfered by host-cell mutations. Applications to coliphage fd, a secretion type phage, are shown as a measurement example. A chimera between fd and a plasmid pBR322 is cultured more than 100 h. The process of population changeovers by deletion mutants indicates that the deletion hot spots exist in this cloning vector and that this apparatus can be used also for testing instability of a recombinant DNA.

Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome

PubMed Central

Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

2015-01-01

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. DOI: http://dx.doi.org/10.7554/eLife.06602.001 PMID:26026149

APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

PubMed Central

Mills, Kate M.; Brocardo, Mariana G.; Henderson, Beric R.

2016-01-01

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. PMID:26658612

Leigh disease with brainstem involvement in complex I deficiency due to assembly factor NDUFAF2 defect.

PubMed

Herzer, M; Koch, J; Prokisch, H; Rodenburg, R; Rauscher, C; Radauer, W; Forstner, R; Pilz, P; Rolinski, B; Freisinger, P; Mayr, J A; Sperl, W

2010-02-01

Mitochondrial NADH: ubiquinone oxidoreductase (complex I) deficiency accounts for most defects in mitochondrial oxidative phosphorylation. Pathogenic mutations have been described in all 7 mitochondrial and 12 of the 38 nuclear encoded subunits as well as in assembly factors by interfering with the building of the mature enzyme complex within the inner mitochondrial membrane. We now describe a male patient with a novel homozygous stop mutation in the NDUFAF2 gene. The boy presented with severe apnoea and nystagmus. MRI showed brainstem lesions without involvement of basal ganglia and thalamus, plasma lactate was normal or close to normal. He died after a fulminate course within 2 months after the first crisis. Neuropathology verified Leigh disease. We give a synopsis with other reported patients. Within the clinical spectrum of Leigh disease, patients with mutations in NDUFAF2 present with a distinct clinical pattern with predominantly brainstem involvement on MRI. The diagnosis should not be missed in spite of the normal lactate and lack of thalamus and basal ganglia changes on brain MRI.

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

PubMed Central

Lehe, Rémi; Hallatschek, Oskar; Peliti, Luca

2012-01-01

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

Assessing pathogenicity for novel mutation/sequence variants: the value of healthy older individuals.

PubMed

Zatz, Mayana; Pavanello, Rita de Cassia M; Lourenço, Naila Cristina V; Cerqueira, Antonia; Lazar, Monize; Vainzof, Mariz

2012-12-01

Improvement in DNA technology is increasingly revealing unexpected/unknown mutations in healthy persons and generating anxiety due to their still unknown health consequences. We report a 44-year-old healthy father of a 10-year-old daughter with bilateral coloboma and hearing loss, but without muscle weakness, in whom a whole-genome CGH revealed a deletion of exons 38-44 in the dystrophin gene. This mutation was inherited from her asymptomatic father, who was further clinically and molecularly evaluated for prognosis and genetic counseling (GC). This deletion was never identified by us in 982 Duchenne/Becker patients. To assess whether the present case represents a rare case of non-penetrance, and aiming to obtain more information for prognosis and GC, we suggested that healthy older relatives submit their DNA for analysis, to which several complied. Mutation analysis revealed that his mother, brother, and 56-year-old maternal uncle also carry the 38-44 deletion, suggesting it an unlikely cause of muscle weakness. Genome sequencing will disclose mutations and variants whose health impact are still unknown, raising important problems in interpreting results, defining prognosis, and discussing GC. We suggest that, in addition to family history, keeping the DNA of older relatives could be very informative, in particular for those interested in having their genome sequenced.

Active-to-absorbing-state phase transition in an evolving population with mutation.

PubMed

Sarkar, Niladri

2015-10-01

We study the active to absorbing phase transition (AAPT) in a simple two-component model system for a species and its mutant. We uncover the nontrivial critical scaling behavior and weak dynamic scaling near the AAPT that shows the significance of mutation and highlights the connection of this model with the well-known directed percolation universality class. Our model should be a useful starting point to study how mutation may affect extinction or survival of a species.

Dysregulated endocardial TGFβ signaling and mesenchymal transformation result in heart outflow tract septation failure.

PubMed

Ma, Mancheong; Li, Peng; Shen, Hua; Estrada, Kristine D; Xu, Jian; Kumar, S Ram; Sucov, Henry M

2016-01-01

Heart outflow tract septation in mouse embryos carrying mutations in retinoic acid receptor genes fails with complete penetrance. In this mutant background, ectopic TGFβ signaling in the distal outflow tract is responsible for septation failure, but it was uncertain what tissue was responsive to ectopic TGFβ and why this response interfered with septation. By combining RAR gene mutation with tissue-specific Cre drivers and a conditional type II TGFβ receptor (Tgfbr2) allele, we determined that ectopic activation of TGFβ signaling in the endocardium is responsible for septation defects. Ectopic TGFβ signaling results in ectopic mesenchymal transformation of the endocardium and thereby in improperly constituted distal OFT cushions. Our analysis highlights the interactions between myocardium, endocardium, and neural crest cells in outflow tract morphogenesis, and demonstrates the requirement for proper TGFβ signaling in outflow tract cushion organization and septation. Copyright © 2015. Published by Elsevier Inc.

Overproduction of Three Genes Leads to Camphor Resistance and Chromosome Condensation in Escherichia Coli

PubMed Central

Hu, K. H.; Liu, E.; Dean, K.; Gingras, M.; DeGraff, W.; Trun, N. J.

1996-01-01

We isolated and characterized three genes, crcA, cspE and crcB, which when present in high copy confer camphor resistance on a cell and suppress mutations in the chromosomal partition gene mukB. Both phenotypes require the same genes. Unlike chromosomal camphor resistant mutants, high copy number crcA, cspE and crcB do not result in an increase in the ploidy of the cells. The cspE gene has been previously identified as a cold shock-like protein with homologues in all organisms tested. We also demonstrate that camphor causes the nucleoids to decondense in vivo and when the three genes are present in high copy, the chromosomes do not decondense. Our results implicate camphor and mukB mutations as interfering with chromosome condensation and high copy crcA, cspE and crcB as promoting or protecting chromosome folding. PMID:8844142

X‐linked retinoschisis: an update

PubMed Central

Sikkink, Stephen K; Biswas, Susmito; Parry, Neil R A; Stanga, Paulo E; Trump, Dorothy

2007-01-01

X‐linked retinoschisis is the leading cause of macular degeneration in males and leads to splitting within the inner retinal layers leading to visual deterioration. Many missense and protein truncating mutations have now been identified in the causative retinoschisis gene (RS1) which encodes a 224 amino acid secreting retinal protein, retinoschisin. Retinoschisin octamerises is implicated in cell–cell interactions and cell adhesion perhaps by interacting with β2 laminin. Mutations cause loss of retinoschisin function by one of the three mechanisms: by interfering with protein secretion, by preventing its octamerisation or by reducing function in the secreted octamerised protein. The development of retinoschisis mouse models have provided a model system that closely resembles the human disease. Recent reports of RS1 gene transfer to these models and the sustained restoration of some retinal function and morphology suggest gene replacement may be a possible future therapy for patients. PMID:17172462

Troyer Syndrome Protein Spartin Is Mono-Ubiquitinated and Functions in EGF Receptor Trafficking

PubMed Central

Jupille, Henri; Fatheddin, Parvin; Puertollano, Rosa

2007-01-01

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia caused by mutation in the spartin (SPG20) gene, which encodes a widely expressed protein of unknown function. This mutation results in premature protein truncation and thus might signify a loss-of-function disease mechanism. In this study, we have found that spartin is mono-ubiquitinated and functions in degradation of the epidermal growth factor receptor (EGFR). Upon EGF stimulation, spartin translocates from the cytoplasm to the plasma membrane and colocalizes with internalized EGF-Alexa. Knockdown of spartin by small interfering RNA decreases the rate of EGFR degradation and also affects EGFR internalization, recycling, or both. Furthermore, overexpression of spartin results in a prominent decrease in EGFR degradation. Taken together, our data suggest that spartin is involved in the intracellular trafficking of EGFR and that impaired endocytosis may underlie the pathogenesis of Troyer syndrome. PMID:17332501

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

PubMed

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

2017-07-01

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

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

NASA Astrophysics Data System (ADS)

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

2017-07-01

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

Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging.

PubMed Central

Donzé, O; Spahr, P F

1992-01-01

The Rous sarcoma virus (RSV) RNA leader sequence carries three open reading frames (uORFs) upstream of the AUG initiator of the gag gene. We studied, in vivo, the role of these uORFs by changing two or three nucleotides of the three AUGs or by deleting the first uORF. Our results show that (i) unlike most previously characterized uORFs, which decrease translation, the first uORF (AUG1) of RSV acts as an enhancer of translation, since absence of the first AUG decreased translation; AUG3 also modulates translation, probably by interfering with scanning ribosomes as described for other upstream ORFs, and mutation of AUG2 had no effect on translation. (ii) Mutation of each of the upstream AUGs lowered the infectivity of progeny virions. (iii) Unexpectedly, mutation of AUG1 and/or AUG3 dramatically reduced RNA packaging by 50-to 100-fold, unlike mutation of AUG2 which did not alter RNA packaging efficiency. Additional mutants in the vicinity of uORF1 and uORF3 were constructed in order to elucidate the mechanism by which uORFs affect RNA packaging: a translation model requiring uORFs 1 and 3, and involving ribosome pausing at AUG 3 is discussed. Images PMID:1327749

Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function

DOE PAGES

Chan, Chun; Fan, Jun; Messer, Andrew E.; ...

2016-04-22

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomersmore » are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. Lastly, these phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.« less

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

PubMed

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Hernández-Laín, Aurelio; Coca-Robinot, David; Rivera, Henry; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, MiguelÁngel; Martínez-Azorín, Francisco

2016-02-29

Whole-exome sequencing (WES) was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase (CK), deficiency of mitochondrial complex III and depletion of mtDNA. With WES data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in Thymidine kinase 2 gene (TK2; NM_004614.4:c.323C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes (MDS). This patient presents an atypical TK2 related-myopathic form of MDS, because despite having a very low content of mtDNA (<20%), she presents a slower and less severe evolution of the disease. In conclusion, our data confirm the role of TK2 gene in MDS and expanded the phenotypic spectrum.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

PubMed

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Rivera, Henry; Hernández-Laín, Aurelio; Coca-Robinot, David; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

2017-01-01

Whole-exome sequencing was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase, deficiency of mitochondrial complex III and depletion of mtDNA. With whole-exome sequencing data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in thymidine kinase 2 gene ( TK2; NM_004614.4:c.323 C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes. This patient presents an atypical TK2-related myopathic form of mtDNA depletion syndromes, because despite having a very low content of mtDNA (<20%), she presents a slower and less severe evolution of the disease. In conclusion, our data confirm the role of TK2 gene in mtDNA depletion syndromes and expanded the phenotypic spectrum.

Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function

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

Chan, Chun; Fan, Jun; Messer, Andrew E.

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomersmore » are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. Lastly, these phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.« less

Rare beneficial mutations can halt Muller's ratchet

NASA Astrophysics Data System (ADS)

Balick, Daniel; Goyal, Sidhartha; Jerison, Elizabeth; Neher, Richard; Shraiman, Boris; Desai, Michael

2012-02-01

In viral, bacterial, and other asexual populations, the vast majority of non-neutral mutations are deleterious. This motivates the application of models without beneficial mutations. Here we show that the presence of surprisingly few compensatory mutations halts fitness decay in these models. Production of deleterious mutations is balanced by purifying selection, stabilizing the fitness distribution. However, stochastic vanishing of fitness classes can lead to slow fitness decay (i.e. Muller's ratchet). For weakly deleterious mutations, production overwhelms purification, rapidly decreasing population fitness. We show that when beneficial mutations are introduced, a stable steady state emerges in the form of a dynamic mutation-selection balance. We argue this state is generic for all mutation rates and population sizes, and is reached as an end state as genomes become saturated by either beneficial or deleterious mutations. Assuming all mutations have the same magnitude selective effect, we calculate the fraction of beneficial mutations necessary to maintain the dynamic balance. This may explain the unexpected maintenance of asexual genomes, as in mitochondria, in the presence of selection. This will affect in the statistics of genetic diversity in these populations.

Late-onset Pompe disease: what is the prevalence of limb-girdle muscular weakness presentation?

PubMed

Lorenzoni, Paulo José; Kay, Cláudia Suemi Kamoi; Higashi, Nádia Sugano; D'Almeida, Vânia; Werneck, Lineu Cesar; Scola, Rosana Herminia

2018-04-01

Pompe disease is an inherited disease caused by acid alpha-glucosidase (GAA) deficiency. A single center observational study aimed at assessing the prevalence of late-onset Pompe disease in a high-risk Brazilian population, using the dried blood spot test to detect GAA deficiency as a main screening tool. Dried blood spots were collected for GAA activity assay from 24 patients with "unexplained" limb-girdle muscular weakness without vacuolar myopathy in their muscle biopsy. Samples with reduced enzyme activity were also investigated for GAA gene mutations. Of the 24 patients with dried blood spots, one patient (4.2%) showed low GAA enzyme activity (NaG/AaGIA: 40.42; %INH: 87.22%). In this patient, genetic analysis confirmed two heterozygous mutations in the GAA gene (c.-32-13T>G/p.Arg854Ter). Our data confirm that clinicians should look for late-onset Pompe disease in patients whose clinical manifestation is an "unexplained" limb-girdle weakness even without vacuolar myopathy in muscle biopsy.

Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts.

PubMed

Müller, Gerd A; Hansen, Uwe; Xu, Zhi; Griswold, Benjamin; Talan, Mark I; McDonnell, Nazli B; Briest, Wilfried

2012-02-01

The vascular type of the Ehlers-Danlos syndrome (vEDS) is caused by dominant-negative mutations in the procollagen type III (COL3A1) gene. Patients with this autosomal dominant disorder have a shortened life expectancy due to complications from ruptured vessels or hollow organs. We tested the effectiveness of allele-specific RNA interference (RNAi) to reduce the mutated phenotype in fibroblasts. Small-interfering RNAs (siRNAs) discriminating between wild-type and mutant COL3A1 allele were identified by a luciferase reporter gene assay and in primary fibroblasts from a normal donor and a patient with vEDS. The best discriminative siRNA with the mutation at position 10 resulted in >90% silencing of the mutant allele without affecting the wild-type allele. Transmission and immunogold electron microscopy of extracted extracellular matrices from untreated fibroblasts of the patient with vEDS revealed structurally abnormal fibrils. After siRNA treatment, collagen fibrils became similar to fibrils from fibroblasts of normal and COL3A1 haploinsufficient donors. In addition, it was shown that expression of mutated COL3A1 activates the unfolded protein response and that reduction of the amount of mutated protein by siRNA reduces cellular stress. Taken together, the results provide evidence that allele-specific siRNAs are able to reduce negative effects of mutated COL3A1 proteins. Thus, the application of allele-specific RNAi may be a promising direction for future personalized therapies to reduce the severity of vEDS.

IARS mutation causes prenatal death in Japanese Black cattle.

PubMed

Hirano, Takashi; Matsuhashi, Tamako; Takeda, Kenji; Hara, Hiromi; Kobayashi, Naohiko; Kita, Kazuo; Sugimoto, Yoshikazu; Hanzawa, Kei

2016-09-01

Isoleucyl-tRNA synthetase (IARS) c.235G > C (p.V79L) is a causative mutation for a recessive disease called IARS disorder in Japanese black cattle. The disease is involved in weak calf syndrome and is characterized by low birth weight, weakness and poor suckling. The gestation period is often slightly extended, implying that intrauterine growth is retarded. In a previous analysis of 2597 artificial insemination (AI) procedures, we suggested that the IARS mutation might contribute toward an increase in the incidence of prenatal death. In this study, we extended this analysis to better clarify the association between the IARS mutation and prenatal death. The IARS genotypes of 92 animals resulting from crosses between carrier (G/C) × G/C were 27 normal (G/G), 55 G/C and 10 affected animals (C/C) (expected numbers: 23, 46 and 23, respectively). Compared to the expected numbers, there were significantly fewer affected animals in this population (P < 0.05), suggesting that more than half of the affected embryos died prenatally. When the number of AI procedures examined was increased to 11 580, the frequency of re-insemination after G/C × G/C insemination was significantly higher at 61-140 days (P < 0.001). The findings suggested that the homozygous IARS mutation not only causes calf death, but also embryonic or fetal death. © 2016 Japanese Society of Animal Science.

Subclinical nonautoimmune hyperthyroidism in a family segregates with a thyrotropin receptor mutation with weakly increased constitutive activity.

PubMed

Nishihara, Eijun; Chen, Chun-Rong; Higashiyama, Takuya; Mizutori-Sasai, Yumiko; Ito, Mitsuru; Kubota, Sumihisa; Amino, Nobuyuki; Miyauchi, Akira; Rapoport, Basil

2010-11-01

Subclinical hyperthyroidism is usually associated with Graves' disease or toxic nodular goiter. Here we report a family with hereditary subclinical hyperthyroidism caused by a constitutively activating germline mutation of the thyrotropin receptor (TSHR) gene. The proband was a 64-year-old Japanese woman who presented with a thyroid nodule and was found to be euthyroid with a suppressed serum TSH. The nodule was not hot. Although antibodies to thyroid peroxidase and thyroglobulin antibodies were present, TSHR antibodies were not detected by TSH-binding inhibition or by bioassay. Two of her middle-aged sons, but not her daughter, also had subclinical hyperthyroidism without TSHR antibodies. Without therapy, the clinical condition of the affected individuals remained unchanged over 3 years without development of overt hyperthyroidism. A novel heterozygous TSHR point mutation causing a glutamic acid to lysine substitution at codon 575 (E575K) in the second extracellular loop was detected in the three family members with subclinical hyperthyroidism, but was absent in her one daughter with normal thyroid function. In vitro functional studies of the E575K TSHR mutation demonstrated a weak, but significant, increase in constitutive activation of the cAMP pathway. Although hereditary nonautoimmune overt hyperthyroidism is very rare, TSHR activating mutations as a cause of subclinical hyperthyroidism may be more common and should be considered in the differential diagnosis, especially if familial.

An exonic missense mutation c.28G>A is associated with weak B blood group by affecting RNA splicing of the ABO gene.

PubMed

Cai, Xiaohong; Qian, Chengrui; Wu, Wenman; Lei, Hang; Ding, Qiulan; Zou, Wei; Xiang, Dong; Wang, Xuefeng

2017-09-01

The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change. Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B 310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B weak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing. While plasma total GTB transfer capacity was undetectable in this B 3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B weak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B 310 cDNA and B cDNA. The mRNA expression level of B 310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells. ABO c.28G>A mutation may cause B 3 -like subgroup by affecting RNA splicing of the ABO gene. © 2017 AABB.

[Clinical features and ETFDH mutations of children with late-onset glutaric aciduria type II: a report of two cases].

PubMed

Cheng, Yan-Yang; Tang, Yue; Liu, Ao-Jie; Wei, Li; Lin, Lan; Zhang, Jing; Zhi, Liang

2017-09-01

To investigate the clinical and genetic features of two families with late-onset glutaric aciduria type II caused by ETFDH mutations. Target gene sequence capture and next generation sequencing were used for sequencing of suspected patients and their family members. The patients' clinical features were retrospectively analyzed and literature review was performed. The probands of the two families had a clinical onset at the ages of 10 years and 5.5 years respectively, with the clinical manifestations of muscle weakness and muscle pain. Laboratory examinations revealed significant increases in the serum levels of creatine kinase, creatine kinase-MB, and lactate dehydrogenase. Tandem mass spectrometry showed increases in various types of acylcarnitines. The analysis of urine organic acids showed an increase in glutaric acid. Electromyography showed myogenic damage in both patients. Gene detection showed two novel mutations in the ETFDH gene (c.1331T>C from the mother and c.824C>T from the father) in patient 1, and the patient's younger brother carried the c.1331T>C mutation but had a normal phenotype. In patient 2, there was a novel mutation (c.177insT from the father) and a known mutation (c.1474T>C from the mother) in the ETFDH gene. Several family members carried such mutations. Both patients were diagnosed with glutaric aciduria type II. Their symptoms were improved after high-dose vitamin B2 treatment. For patients with unexplained muscle weakness and pain, serum creatine kinase, acylcarnitines, and urinary organic acids should be measured, and the possibility of glutaric aciduria type II should be considered. Genetic detection is helpful to make a confirmed diagnosis.

A Middle Eastern Founder Mutation Expands the Genotypic and Phenotypic Spectrum of Mitochondrial MICU1 Deficiency: A Report of 13 Patients.

PubMed

Musa, Sara; Eyaid, Wafaa; Kamer, Kimberli; Ali, Rehab; Al-Mureikhi, Mariam; Shahbeck, Noora; Al Mesaifri, Fatma; Makhseed, Nawal; Mohamed, Zakkiriah; AlShehhi, Wafaa Ali; Mootha, Vamsi K; Juusola, Jane; Ben-Omran, Tawfeg

2018-05-03

MICU1 encodes a Ca 2+ sensing, regulatory subunit of the mitochondrial uniporter, a selective calcium channel within the organelle's inner membrane. Ca 2+ entry into mitochondria helps to buffer cytosolic Ca 2+ transients and also activates ATP production within the organelle. Mutations in MICU1 have previously been reported in 17 children from nine families with muscle weakness, fatigue, normal lactate, and persistently elevated creatine kinase, as well as variable features that include progressive extrapyramidal signs, learning disabilities, nystagmus, and cataracts. In this study, we report the clinical features of an additional 13 patients from consanguineous Middle Eastern families with recessive mutations in MICU1. Of these patients, 12/13 are homozygous for a novel founder mutation c.553C>T (p.Q185*) that is predicted to lead to a complete loss of function of MICU1, while one patient is compound heterozygous for this mutation and an intragenic duplication of exons 9 and 10. The founder mutation occurs with a minor allele frequency of 1:60,000 in the ExAC database, but in ~1:500 individual in the Middle East. All 13 of these patients presented with developmental delay, learning disability, muscle weakness and easy fatigability, and failure to thrive, as well as additional variable features we tabulate. Consistent with previous cases, all of these patients had persistently elevated serum creatine kinase with normal lactate levels, but they also exhibited elevated transaminase enzymes. Our work helps to better define the clinical sequelae of MICU1 deficiency. Furthermore, our work suggests that targeted analysis of the MICU1 founder mutation in Middle Eastern patients may be warranted.

Work ability in patients with polymyositis and dermatomyositis: An explorative and descriptive study.

PubMed

Regardt, Malin; Welin Henriksson, Elisabet; Sandqvist, Jan; Lundberg, Ingrid E; Schult, Marie-Louise

2015-01-01

Polymyositis (PM) and dermatomyositis (DM) are rare, chronic inflammatory diseases leading to muscle weakness and low muscle endurance. The muscle weakness may lead to restrictions in daily activities and low health-related quality of life. This study aimed to investigate the work situation, work ability, work-related risk factors, and influence of the physical and psycho-social work environment in patients with PM and DM. Patients with PM/DM were assessed using the Work Ability Index (WAI), and the Work Environment Impact Scale (WEIS). Forty-eight patients (PM n = 25 and DM n = 23) participated (women/men: 29/19) with a mean age of 54 years (range 28-67 years, SD.10) and mean disease duration of nine years (SD.9). Forty-four percent worked full-time, 31% part-time and 25% were on full-time sick leave. More than 50% self-rated work ability as "poor" or "less good". Physically strenuous work components were present "quite to very often" in 23-79% and more in patients on sick leave ≥ 2 years. For those working, the interfering factors in the work environment concerned task and time demands. Supporting factors concerned meaning of work, interactions with co-workers and others. Self-rated work ability correlated moderately-highly positive with percentage of full-time employment, work-related risk factors and opportunities and constraints in the work environment. Poor self-rated work ability is common in patients with PM/DM indicating a need to identify interfering risk factors and support patients to enhance work performance.

PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality.

PubMed

Dehouck, Yves; Kwasigroch, Jean Marc; Gilis, Dimitri; Rooman, Marianne

2011-05-13

The rational design of modified proteins with controlled stability is of extreme importance in a whole range of applications, notably in the biotechnological and environmental areas, where proteins are used for their catalytic or other functional activities. Future breakthroughs in medical research may also be expected from an improved understanding of the effect of naturally occurring disease-causing mutations on the molecular level. PoPMuSiC-2.1 is a web server that predicts the thermodynamic stability changes caused by single site mutations in proteins, using a linear combination of statistical potentials whose coefficients depend on the solvent accessibility of the mutated residue. PoPMuSiC presents good prediction performances (correlation coefficient of 0.8 between predicted and measured stability changes, in cross validation, after exclusion of 10% outliers). It is moreover very fast, allowing the prediction of the stability changes resulting from all possible mutations in a medium size protein in less than a minute. This unique functionality is user-friendly implemented in PoPMuSiC and is particularly easy to exploit. Another new functionality of our server concerns the estimation of the optimality of each amino acid in the sequence, with respect to the stability of the structure. It may be used to detect structural weaknesses, i.e. clusters of non-optimal residues, which represent particularly interesting sites for introducing targeted mutations. This sequence optimality data is also expected to have significant implications in the prediction and the analysis of particular structural or functional protein regions. To illustrate the interest of this new functionality, we apply it to a dataset of known catalytic sites, and show that a much larger than average concentration of structural weaknesses is detected, quantifying how these sites have been optimized for function rather than stability. The freely available PoPMuSiC-2.1 web server is highly useful for identifying very rapidly a list of possibly relevant mutations with the desired stability properties, on which subsequent experimental studies can be focused. It can also be used to detect sequence regions corresponding to structural weaknesses, which could be functionally important or structurally delicate regions, with obvious applications in rational protein design.

A heterozygous 21-bp deletion in CAPN3 causes dominantly inherited limb girdle muscular dystrophy.

PubMed

Vissing, John; Barresi, Rita; Witting, Nanna; Van Ghelue, Marijke; Gammelgaard, Lise; Bindoff, Laurence A; Straub, Volker; Lochmüller, Hanns; Hudson, Judith; Wahl, Christoph M; Arnardottir, Snjolaug; Dahlbom, Kathe; Jonsrud, Christoffer; Duno, Morten

2016-08-01

Limb girdle muscular dystrophy type 2A is the most common limb girdle muscular dystrophy form worldwide. Although strict recessive inheritance is assumed, patients carrying a single mutation in the calpain 3 gene (CAPN3) are reported. Such findings are commonly attributed to incomplete mutation screening. In this investigation, we report 37 individuals (age range: 21-85 years, 21 females and 16 males) from 10 families in whom only one mutation in CAPN3 could be identified; a 21-bp, in-frame deletion (c.643_663del21). This mutation co-segregated with evidence of muscle disease and autosomal dominant transmission in several generations. Evidence of muscle disease was indicated by muscle pain, muscle weakness and wasting, significant fat replacement of muscles on imaging, myopathic changes on muscle biopsy and loss of calpain 3 protein on western blotting. Thirty-one of 34 patients had elevated creatine kinase or myoglobin. Muscle weakness was generally milder than observed in limb girdle muscular dystrophy type 2A, but affected the same muscle groups (proximal leg, lumbar paraspinal and medial gastrocnemius muscles). In some cases, the weakness was severely disabling. The 21-bp deletion did not affect mRNA maturation. Calpain 3 expression in muscle, assessed by western blot, was below 15% of normal levels in the nine mutation carriers in whom this could be tested. Haplotype analysis in four families from three different countries suggests that the 21-bp deletion is a founder mutation. This study provides strong evidence that heterozygosity for the c.643_663del21 deletion in CAPN3 results in a dominantly inherited muscle disease. The normal expression of mutated mRNA and the severe loss of calpain 3 on western blotting, suggest a dominant negative effect with a loss-of-function mechanism affecting the calpain 3 homodimer. This renders patients deficient in calpain 3 as in limb girdle muscular dystrophy type 2A, albeit in a milder form in most cases. Based on findings in 10 families, our study indicates that a dominantly inherited pattern of calpainopathy exists, and should be considered in the diagnostic work-up and genetic counselling of patients with calpainopathy and single-allele aberrations in CAPN3. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

CRISPR-mediated targeting of HER2 inhibits cell proliferation through a dominant negative mutation.

PubMed

Wang, Huajing; Sun, William

2017-01-28

With the discovery of the CRISPR/Cas9 technology, genome editing could be performed in a rapid, precise and effective manner. Its potential applications in functional interrogation of cancer-causing genes and cancer therapy have been extensively explored. In this study, we demonstrated the use of the CRISPR/Cas9 system to directly target the oncogene HER2. Directing Cas9 to exons of the HER2 gene inhibited cell growth in breast cancer cell lines that harbor amplification of the HER2 locus. The inhibitory effect was potentiated with the addition of PARP inhibitors. Unexpectedly, CRISPR-induced mutations did not significantly affect the level of HER2 protein expression. Instead, CRISPR targeting appeared to exert its effect through a dominant negative mutation. This HER2 mutant interfered with the MAPK/ERK axis of HER2 downstream signaling. Our work provides a novel mechanism underlying the anti-cancer effects of HER2-targeting by CRISPR/Cas9, which is distinct from the clinical drug Herceptin. In addition, it opens up the possibility that incomplete CRISPR targeting of certain oncogenes could still have therapeutic value by generation of dominant negative mutants. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

PubMed

Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

2005-08-15

Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

Clinical features of the myasthenic syndrome arising from mutations in GMPPB.

PubMed

Rodríguez Cruz, Pedro M; Belaya, Katsiaryna; Basiri, Keivan; Sedghi, Maryam; Farrugia, Maria Elena; Holton, Janice L; Liu, Wei Wei; Maxwell, Susan; Petty, Richard; Walls, Timothy J; Kennett, Robin; Pitt, Matthew; Sarkozy, Anna; Parton, Matt; Lochmüller, Hanns; Muntoni, Francesco; Palace, Jacqueline; Beeson, David

2016-08-01

Congenital myasthenic syndrome (CMS) due to mutations in GMPPB has recently been reported confirming the importance of glycosylation for the integrity of neuromuscular transmission. Review of case notes of patients with mutations in GMPPB to identify the associated clinical, neurophysiological, pathological and laboratory features. In addition, serum creatine kinase (CK) levels within the Oxford CMS cohort were retrospectively analysed to assess its usefulness in the differential diagnosis of this new entity. All patients had prominent limb-girdle weakness with minimal or absent craniobulbar manifestations. Presentation was delayed beyond infancy with proximal muscle weakness and most patients recall poor performance in sports during childhood. Neurophysiology showed abnormal neuromuscular transmission only in the affected muscles and myopathic changes. Muscle biopsy showed dystrophic features and reduced α-dystroglycan glycosylation. In addition, myopathic changes were present on muscle MRI. CK was significantly increased in serum compared to other CMS subtypes. Patients were responsive to pyridostigimine alone or combined with 3,4-diaminopyridine and/or salbutamol. Patients with GMPPB-CMS have phenotypic features aligned with CMS subtypes harbouring mutations within the early stages of the glycosylation pathway. Additional features shared with the dystroglycanopathies include myopathic features, raised CK levels and variable mild cognitive delay. This syndrome underlines that CMS can occur in the absence of classic myasthenic manifestations such as ptosis and ophthalmoplegia or facial weakness, and links myasthenic disorders with dystroglycanopathies. This report should facilitate the recognition of this disorder, which is likely to be underdiagnosed and can benefit from symptomatic treatment. 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/

GNE Myopathy in Turkish Sisters with a Novel Homozygous Mutation

PubMed Central

Diniz, Gulden; Secil, Yaprak; Ceylaner, Serdar; Tokucoglu, Figen; Türe, Sabiha; Celebisoy, Mehmet; İncesu, Tülay Kurt; Akhan, Galip

2016-01-01

Background. Hereditary inclusion body myopathy is caused by biallelic defects in the GNE gene located on chromosome 9p13. It generally affects adults older than 20 years of age. Methods and Results. In this study, we present two Turkish sisters with progressive myopathy and describe a novel mutation in the GNE gene. Both sisters had slightly higher levels of creatine kinase (CK) and muscle weakness. The older sister presented at 38 years of age with an inability to climb steps, weakness, and a steppage gait. Her younger sister was 36 years old and had similar symptoms. The first symptoms of the disorder were seen when the sisters were 30 and 34 years old, respectively. The muscle biopsy showed primary myopathic features and presence of rimmed vacuoles. DNA analysis demonstrated the presence of previously unknown homozygous mutations [c.2152 G>A (p.A718T)] in the GNE genes. Conclusion. Based on our literature survey, we believe that ours is the first confirmed case of primary GNE myopathy with a novel missense mutation in Turkey. These patients illustrate that the muscle biopsy is still an important method for the differential diagnosis of vacuolar myopathies in that the detection of inclusions is required for the definitive diagnosis. PMID:27298745

A case of adult-onset reducing body myopathy presenting a novel clinical feature, asymmetrical involvement of the sternocleidomastoid and trapezius muscles.

PubMed

Fujii, Takayuki; Hayashi, Shintaro; Kawamura, Nobutoshi; Higuchi, Masa-Aki; Tsugawa, Jun; Ohyagi, Yasumasa; Hayashi, Yukiko K; Nishino, Ichizo; Kira, Jun-Ichi

2014-08-15

We herein report a 32-year-old woman with adult-onset reducing body myopathy (RBM) who had a mutation in the four-and-a-half LIM domain 1 gene (FHL1) and showed a marked asymmetrical involvement of sternocleidomastoid and trapezius muscles. At 30 years of age she noticed bilateral foot drop, and over the next two years developed difficulty raising her right arm. At 32 years of age she was admitted to our hospital for a diagnostic evaluation. Neurological examination showed moderate weakness and atrophy of her right sternocleidomastoid muscle, right trapezius muscle, and bilateral upper proximal muscles. There were severe weakness and atrophy of her bilateral tibialis anterior muscles. Her deep tendon reflexes were hypoactive in her upper extremities. Her serum creatine kinase level was mildly increased. Muscle biopsy specimens from the left tibialis anterior muscle revealed marked variation in fiber size, some necrotic or regenerating fibers, and reducing bodies. Gene analysis of FHL1 demonstrated a mutation: a heterozygous missense mutation of c.377G>A (p. C126T) in FHL1. Compared with previous adult-onset RBM cases harboring mutations in FHL1, our case was characterized by asymmetrical atrophy of the sternocleidomastoid and trapezius muscles. Copyright © 2014 Elsevier B.V. All rights reserved.

[Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is caused by a mutation in TFG].

PubMed

Ishiura, Hiroyuki; Tsuji, Shoji

2013-01-01

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal dominant neurodegenerative disease characterized by proximal predominant weakness and muscle atrophy accompanied by distal sensory disturbance. Linkage analysis using 4 families identified a region on chromosome 3 showing a LOD score exceeding 4. Further refinement of candidate region was performed by haplotype analysis using high-density SNP data, resulting in a minimum candidate region spanning 3.3 Mb. Exome analysis of an HMSN-P patient revealed a mutation (c.854C>T, p.Pro285Leu) in TRK-fused gene (TFG). The identical mutation was found in the four families, which cosegregated with the disease. The mutation was neither found in Japanese control subjects nor public databases. Detailed haplotype analysis suggested two independent origins of the mutation. These findings indicate that the mutation in TFG causes HMSN-P.

Leigh syndrome associated with a novel mutation in the COX15 gene.

PubMed

Miryounesi, Mohammad; Fardaei, Majid; Tabei, Seyed Mohammadbagher; Ghafouri-Fard, Soudeh

2016-06-01

Leigh syndrome (LS) is a subacute necrotizing encephalomyelopathy with a diverse range of symptoms, such as psychomotor delay or regression, weakness, hypotonia, truncal ataxia, intention tremor as well as lactic acidosis in the blood, cerebrospinal fluid or urine. Both nuclear gene defects and mutations of the mitochondrial genome have been detected in these patients. Here we report a 7-year-old girl with hypotonia, tremor, developmental delay and psychomotor regression. However, serum lactate level as well as brain magnetic resonance imaging were normal. Mutational analysis has revealed a novel mutation in exon 4 of COX15 gene (c.415C>G) which results in p.Leu139Val. Previous studies have demonstrated that COX15 mutations are associated with typical LS as well as fatal infantile hypertrophic cardiomyopathy. Consequently, clinical manifestations of COX15 mutations may be significantly different in patients. Such information is of practical importance in genetic counseling.

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

PubMed

Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

2016-06-01

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

SPG20 Protein Spartin Is Recruited to Midbodies by ESCRT-III Protein Ist1 and Participates in Cytokinesis

PubMed Central

Renvoisé, Benoît; Parker, Rell L.; Yang, Dong; Bakowska, Joanna C.; Hurley, James H.

2010-01-01

Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1–7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin–Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis. PMID:20719964

Two-Color Coherent Control of Femtosecond Above-Threshold Photoemission from a Tungsten Nanotip.

PubMed

Förster, Michael; Paschen, Timo; Krüger, Michael; Lemell, Christoph; Wachter, Georg; Libisch, Florian; Madlener, Thomas; Burgdörfer, Joachim; Hommelhoff, Peter

2016-11-18

We demonstrate coherent control of multiphoton and above-threshold photoemission from a single solid-state nanoemitter driven by a fundamental and a weak second harmonic laser pulse. Depending on the relative phase of the two pulses, electron emission is modulated with a contrast of the oscillating current signal of up to 94%. Electron spectra reveal that all observed photon orders are affected simultaneously and similarly. We confirm that photoemission takes place within 10 fs. Accompanying simulations indicate that the current modulation with its large contrast results from two interfering quantum pathways leading to electron emission.

Recurrent Muscle Weakness with Rhabdomyolysis, Metabolic Crises, and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations

PubMed Central

Lalani, Seema R.; Liu, Pengfei; Rosenfeld, Jill A.; Watkin, Levi B.; Chiang, Theodore; Leduc, Magalie S.; Zhu, Wenmiao; Ding, Yan; Pan, Shujuan; Vetrini, Francesco; Miyake, Christina Y.; Shinawi, Marwan; Gambin, Tomasz; Eldomery, Mohammad K.; Akdemir, Zeynep Hande Coban; Emrick, Lisa; Wilnai, Yael; Schelley, Susan; Koenig, Mary Kay; Memon, Nada; Farach, Laura S.; Coe, Bradley P.; Azamian, Mahshid; Hernandez, Patricia; Zapata, Gladys; Jhangiani, Shalini N.; Muzny, Donna M.; Lotze, Timothy; Clark, Gary; Wilfong, Angus; Northrup, Hope; Adesina, Adekunle; Bacino, Carlos A.; Scaglia, Fernando; Bonnen, Penelope E.; Crosson, Jane; Duis, Jessica; Maegawa, Gustavo H.B.; Coman, David; Inwood, Anita; McGill, Jim; Boerwinkle, Eric; Graham, Brett; Beaudet, Art; Eng, Christine M.; Hanchard, Neil A.; Xia, Fan; Orange, Jordan S.; Gibbs, Richard A.; Lupski, James R.; Yang, Yaping

2016-01-01

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3–9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3–9. Additionally, a homozygous exons 4–6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3–9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations. PMID:26805781

R521C and R521H mutations in FUS result in weak binding with Karyopherinβ2 leading to Amyotrophic lateral sclerosis: a molecular docking and dynamics study.

PubMed

Swetha, Rayapadi G; Ramaiah, Sudha; Anbarasu, Anand

2017-08-01

Fused in sarcoma (FUS) gene encodes the RNA binding protein FUS. This gene is mapped to chromosome 16p11.2. The FUS protein binds with karyopherineβ2 (Kapβ2) through its proline/tyrosine nuclear localization signal (PY-NLS) that helps in the localization of FUS protein within the nucleus. Arginine residue in 521 position (R521) of PY-NLS plays a vital role in the binding of FUS protein with Kapβ2. Mutations in this position (R521C and R521H) are the most predominant mutations associated with amyotrophic lateral sclerosis (ALS). However, the mechanism by which these mutations lead to ALS is poorly understood. We examined the binding behaviour of the mutants FUS (R521C) and FUS (R521H) with Kapβ2 through protein-protein docking and molecular dynamics simulation. The binding patterns of mutants were compared with the binding behaviour of wild FUS-Kapβ2. Our results suggest that these mutants have relatively weak binding affinity with Kapβ2 when compared with wild FUS-Kapβ2 as indicated by the lesser number of interactions found between the mutant FUS and Kapβ2. Hence, these mutations weakens the binding and this results in the cytoplasmic mislocalization of mutant FUS; and thereby it increases the severity of ALS.

Subclinical Nonautoimmune Hyperthyroidism in a Family Segregates with a Thyrotropin Receptor Mutation with Weakly Increased Constitutive Activity

PubMed Central

Chen, Chun-Rong; Higashiyama, Takuya; Mizutori-Sasai, Yumiko; Ito, Mitsuru; Kubota, Sumihisa; Amino, Nobuyuki; Miyauchi, Akira; Rapoport, Basil

2010-01-01

Background Subclinical hyperthyroidism is usually associated with Graves' disease or toxic nodular goiter. Here we report a family with hereditary subclinical hyperthyroidism caused by a constitutively activating germline mutation of the thyrotropin receptor (TSHR) gene. Methods The proband was a 64-year-old Japanese woman who presented with a thyroid nodule and was found to be euthyroid with a suppressed serum TSH. The nodule was not hot. Although antibodies to thyroid peroxidase and thyroglobulin antibodies were present, TSHR antibodies were not detected by TSH-binding inhibition or by bioassay. Two of her middle-aged sons, but not her daughter, also had subclinical hyperthyroidism without TSHR antibodies. Without therapy, the clinical condition of the affected individuals remained unchanged over 3 years without development of overt hyperthyroidism. Results A novel heterozygous TSHR point mutation causing a glutamic acid to lysine substitution at codon 575 (E575K) in the second extracellular loop was detected in the three family members with subclinical hyperthyroidism, but was absent in her one daughter with normal thyroid function. In vitro functional studies of the E575K TSHR mutation demonstrated a weak, but significant, increase in constitutive activation of the cAMP pathway. Conclusion Although hereditary nonautoimmune overt hyperthyroidism is very rare, TSHR activating mutations as a cause of subclinical hyperthyroidism may be more common and should be considered in the differential diagnosis, especially if familial. PMID:20929407

Severe sensory neuropathy in patients with adult-onset multiple acyl-CoA dehydrogenase deficiency.

PubMed

Wang, Zhaoxia; Hong, Daojun; Zhang, Wei; Li, Wurong; Shi, Xin; Zhao, Danhua; Yang, Xu; Lv, He; Yuan, Yun

2016-02-01

Multiple Acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder of fatty acid oxidation. Most patients with late-onset MADD are clinically characterized by lipid storage myopathy with dramatic responsiveness to riboflavin treatment. Abnormalities of peripheral neuropathy have rarely been reported in patients with late-onset MADD. We describe six patients who presented with proximal limb weakness and loss of sensation in the distal limbs. Muscle biopsy revealed typical myopathological patterns of lipid storage myopathy and blood acylcarnitine profiles showed a combined elevation of multiple acylcarnitines supporting the diagnosis of MADD. However, nerve conduction investigations and sural nerve biopsies in these patients indicated severe axonal sensory neuropathy. Causative ETFDH gene mutations were found in all six cases. No other causative gene mutations were identified in mitochondrial DNA and genes associated with hereditary neuropathies through next-generation-sequencing panel. Late-onset patients with ETFDH mutations can present with proximal muscle weakness and distal sensory neuropathy, which might be a new phenotypic variation, but the precise underlying pathogenesis remains to be elucidated. Copyright © 2015. Published by Elsevier B.V.

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

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

Xu, Fei; Li, Hongling; Sun, Yong, E-mail: sunfanqi2010@163.com

2014-11-28

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

Staying green postharvest: how three mutations in the Arabidopsis chlorophyll b reductase gene NYC1 delay degreening by distinct mechanisms.

PubMed

Jibran, Rubina; Sullivan, Kerry L; Crowhurst, Ross; Erridge, Zoe A; Chagné, David; McLachlan, Andrew R G; Brummell, David A; Dijkwel, Paul P; Hunter, Donald A

2015-11-01

Stresses such as energy deprivation, wounding and water-supply disruption often contribute to rapid deterioration of harvested tissues. To uncover the genetic regulation behind such stresses, a simple assessment system was used to detect senescence mutants in conjunction with two rapid mapping techniques to identify the causal mutations. To demonstrate the power of this approach, immature inflorescences of Arabidopsis plants that contained ethyl methanesulfonate-induced lesions were detached and screened for altered timing of dark-induced senescence. Numerous mutant lines displaying accelerated or delayed timing of senescence relative to wild type were discovered. The underlying mutations in three of these were identified using High Resolution Melting analysis to map to a chromosomal arm followed by a whole-genome sequencing-based mapping method, termed 'Needle in the K-Stack', to identify the causal lesions. All three mutations were single base pair changes and occurred in the same gene, NON-YELLOW COLORING1 (NYC1), a chlorophyll b reductase of the short-chain dehydrogenase/reductase (SDR) superfamily. This was consistent with the mutants preferentially retaining chlorophyll b, although substantial amounts of chlorophyll b were still lost. The single base pair mutations disrupted NYC1 function by three distinct mechanisms, one by producing a termination codon, the second by interfering with correct intron splicing and the third by replacing a highly conserved proline with a non-equivalent serine residue. This non-synonymous amino acid change, which occurred in the NADPH binding domain of NYC1, is the first example of such a mutation in an SDR protein inhibiting a physiological response in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

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

Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

2014-06-15

Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutatedmore » (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.« less

Skeletal muscle weakness in osteogenesis imperfecta mice.

PubMed

Gentry, Bettina A; Ferreira, J Andries; McCambridge, Amanda J; Brown, Marybeth; Phillips, Charlotte L

2010-09-01

Exercise intolerance, muscle fatigue and weakness are often-reported, little-investigated concerns of patients with osteogenesis imperfecta (OI). OI is a heritable connective tissue disorder hallmarked by bone fragility resulting primarily from dominant mutations in the proα1(I) or proα2(I) collagen genes and the recently discovered recessive mutations in post-translational modifying proteins of type I collagen. In this study we examined the soleus (S), plantaris (P), gastrocnemius (G), tibialis anterior (TA) and quadriceps (Q) muscles of mice expressing mild (+/oim) and moderately severe (oim/oim) OI for evidence of inherent muscle pathology. In particular, muscle weight, fiber cross-sectional area (CSA), fiber type, fiber histomorphology, fibrillar collagen content, absolute, relative and specific peak tetanic force (P(o), P(o)/mg and P(o)/CSA respectively) of individual muscles were evaluated. Oim/oim mouse muscles were generally smaller, contained less fibrillar collagen, had decreased P(o) and an inability to sustain P(o) for the 300-ms testing duration for specific muscles; +/oim mice had a similar but milder skeletal muscle phenotype. +/oim mice had mild weakness of specific muscles but were less affected than their oim/oim counterparts which demonstrated readily apparent skeletal muscle pathology. Therefore muscle weakness in oim mice reflects inherent skeletal muscle pathology. Copyright © 2010 Elsevier B.V. All rights reserved.

Natural disease history of the dy2J mouse model of laminin α2 (merosin)-deficient congenital muscular dystrophy.

PubMed

Pasteuning-Vuhman, S; Putker, K; Tanganyika-de Winter, C L; Boertje-van der Meulen, J W; van Vliet, L; Overzier, M; Plomp, J J; Aartsma-Rus, A; van Putten, M

2018-01-01

Merosin deficient congenital muscular dystrophy 1A (MDC1A) is a very rare autosomal recessive disorder caused by mutations in the LAMA2 gene leading to severe and progressive muscle weakness and atrophy. Although over 350 causative mutations have been identified for MDC1A, no treatment is yet available. There are many therapeutic approaches in development, but the lack of natural history data of the mouse model and standardized outcome measures makes it difficult to transit these pre-clinical findings to clinical trials. Therefore, in the present study, we collected natural history data and assessed pre-clinical outcome measures for the dy2J/dy2J mouse model using standardized operating procedures available from the TREAT-NMD Alliance. Wild type and dy2J/dy2J mice were subjected to five different functional tests from the age of four to 32 weeks. Non-tested control groups were taken along to assess whether the functional test regime interfered with muscle pathology. Respiratory function, body weights and creatine kinase levels were recorded. Lastly, skeletal muscles were collected for further histopathological and gene expression analyses. Muscle function of dy2J/dy2J mice was severely impaired at four weeks of age and all mice lost the ability to use their hind limbs. Moreover, respiratory function was altered in dy2J/dy2J mice. Interestingly, the respiration rate was decreased and declined with age, whereas the respiration amplitude was increased in dy2J/dy2J mice when compared to wild type mice. Creatine kinase levels were comparable to wild type mice. Muscle histopathology and gene expression analysis revealed that there was a specific regional distribution pattern of muscle damage in dy2J/dy2J mice. Gastrocnemius appeared to be the most severely affected muscle with a high proportion of atrophic fibers, increased fibrosis and inflammation. By contrast, triceps was affected moderately and diaphragm only mildly. Our study presents a complete natural history dataset which can be used in setting up standardized studies in dy2J/dy2J mice.

[Leigh syndrome resulting from a de novo mitochondrial DNA mutation (T8993G)].

PubMed

Playán, A; Solano-Palacios, A; González de la Rosa, J B; Merino-Arribas, J M; Andreu, A L; López-Pérez, M; Montoya, J

Several degenerative neurological diseases are caused by mutations in the mitochondrial gene coding for subunit 6 of the ATPase. Thus, NARP (neurogenic weakness, ataxia, and retinitis pigmentosa) and Leigh syndromes are associated to a T8993G mutation when the percentage of mutant mitochondrial DNA is low (60 90%) or high (>90%), respectively. Leigh syndrome is also caused by a second mutation in the same position T8993C. The patient, a boy that died at 6 months, had generalized hypotonia, psychomotor delay, hepatomegaly, choreic movements and hyporreflexia. MRI showed hypodensities in the basal ganglia and brain stem as well as hyperlactacidemia. Molecular genetic analysis of the mitochondrial DNA showed that the patient had the T8993G mutation in a percentage higher than 95%. No mutated DNA was detected in blood of the proband s mother, maternal aunt and grandmother. The point mutation T8993G may occur de novo, at high levels, causing neurodegenerative diseases.

Hereditary motor and sensory neuropathy Lom type in a Serbian family.

PubMed

Dacković, J; Keckarević-Marković, M; Komazec, Z; Rakocević-Stojanović, V; Lavrnić, D; Stević, Z; Ribarić, K; Romac, S; Apostolski, S

2008-10-01

Hereditary motor and sensory neuropathy Lom type (HMSNL), also called CMT 4D, a hereditary autosomal recessive neuropathy, caused by mutation in N-Myc downstream regulated gene 1 (NDRG1 gene), was first described in a Bulgarian Gypsy population near Lom and later has been found in Gypsy communities in Italy, Spain, Slovenia and Hungary. We present two siblings with HMSNL, female and male, aged 30 and 26, respectively in a Serbian non-consanguineous family of Gypsy ethnic origin. They had normal developmental milestones. Both had symptoms of lower limb muscle weakness and walking difficulties with frequent falls, which began at the age of seven. At the age of 12, they developed hearing problems and at the age of 15 hand muscle weakness. Neurological examination revealed sensorineural hearing loss, dysarthria, severe distal and mild proximal muscle wasting and weakness, areflexia and impairment of all sensory modalities of distal distribution. Electrophysiological study revealed denervation with severe and early axonal loss. Sensorineural hearing loss was confirmed on electrocochleography and brainstem evoked potentials. Molecular genetic testing confirmed homozygote C564t (R148X) mutation in NDRG1 gene.

Time-series oligonucleotide count to assign antiviral siRNAs with long utility fit in the big data era.

PubMed

Wada, K; Wada, Y; Iwasaki, Y; Ikemura, T

2017-10-01

Oligonucleotides are key elements of nucleic acid therapeutics such as small interfering RNAs (siRNAs). Influenza and Ebolaviruses are zoonotic RNA viruses mutating very rapidly, and their sequence changes must be characterized intensively to design therapeutic oligonucleotides with long utility. Focusing on a total of 182 experimentally validated siRNAs for influenza A, B and Ebolaviruses compiled by the siRNA database, we conducted time-series analyses of occurrences of siRNA targets in these viral genomes. Reflecting their high mutation rates, occurrences of target oligonucleotides evidently fluctuate in viral populations and often disappear. Time-series analysis of the one-base changed sequences derived from each original target identified the oligonucleotide that shows a compensatory increase and will potentially become the 'awaiting-type oligonucleotide'; the combined use of this oligonucleotide with the original can provide therapeutics with long utility. This strategy is also useful for assigning diagnostic reverse transcription-PCR primers with long utility.

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

PubMed Central

Sierant, Malgorzata; Paduszynska, Alina; Kazmierczak-Baranska, Julia; Nacmias, Benedetta; Sorbi, Sandro; Bagnoli, Silvia; Sochacka, Elzbieta; Nawrot, Barbara

2011-01-01

RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide. PMID:21559198

Time-series oligonucleotide count to assign antiviral siRNAs with long utility fit in the big data era

PubMed Central

Wada, K; Wada, Y; Iwasaki, Y; Ikemura, T

2017-01-01

Oligonucleotides are key elements of nucleic acid therapeutics such as small interfering RNAs (siRNAs). Influenza and Ebolaviruses are zoonotic RNA viruses mutating very rapidly, and their sequence changes must be characterized intensively to design therapeutic oligonucleotides with long utility. Focusing on a total of 182 experimentally validated siRNAs for influenza A, B and Ebolaviruses compiled by the siRNA database, we conducted time-series analyses of occurrences of siRNA targets in these viral genomes. Reflecting their high mutation rates, occurrences of target oligonucleotides evidently fluctuate in viral populations and often disappear. Time-series analysis of the one-base changed sequences derived from each original target identified the oligonucleotide that shows a compensatory increase and will potentially become the ‘awaiting-type oligonucleotide’ the combined use of this oligonucleotide with the original can provide therapeutics with long utility. This strategy is also useful for assigning diagnostic reverse transcription-PCR primers with long utility. PMID:28905886

Optical observation of correlated motions in dihydrofolate reductase

NASA Astrophysics Data System (ADS)

Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

2015-03-01

Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

Isolated case of mental retardation and ataxia due to a de novo mitochondrial T8993G mutation

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

De Coo, I.F.M.; Smeets, H.J.M.; Oost, B.A. van

1996-03-01

Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and subacute necrotizing encephalomyelopathy (Leigh disease) are both associated with an alteration of nt 8993 in the mitochondrial ATPase 6 gene. In NARP, the T-to-G transversion at that position changes leucine into arginine. In Leigh syndrome, the same mutation can be found, as can a T-to-C transition, which changes this leucine into proline. Clinical manifestations occur for NARP when {approximately}60%-90% mutated mtDNA is present. In case of Leigh, these percentages usually exceed 95%. It is known that this mutation can segregate very rapidly within pedigrees. Here we report on a sporadic casemore » with mental retardation and ataxia without retinitis pigmentosa in which the T8993G mutation was found. 13 refs., 1 fig.« less

A new de novo missense mutation in MYH2 expands clinical and genetic findings in hereditary myosin myopathies.

PubMed

D'Amico, A; Fattori, F; Bellacchio, E; Catteruccia, M; Servidei, S; Bertini, E

2013-05-01

Congenital myopathy related to mutations in myosin MyHC IIa gene (MYH2) is a rare neuromuscular disease. A single dominant missense mutation has been reported so far in a family in which the affected members had congenital joint contractures at birth, external ophthalmoplegia and proximal muscle weakness. Afterward only additional 4 recessive mutations have been identified in 5 patients presenting a mild non-progressive early-onset myopathy associated with ophthalmoparesis. We report a new de novo MYH2 missense mutation in a baby affected by a congenital myopathy characterized by severe dysphagia, respiratory distress at birth and external ophthalmoplegia. We describe clinical, histopathological and muscle imaging findings expanding the clinical and genetic spectrum of MYH2-related myopathy. Copyright © 2013 Elsevier B.V. All rights reserved.

Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2.

PubMed

Witting, N; Duno, M; Vissing, J

2013-01-01

Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest that this unusual phenotype is caused by translation re-initiation downstream from the mutation site. Copyright © 2012 Elsevier B.V. All rights reserved.

Myotilinopathy in a family with late onset myopathy.

PubMed

Pénisson-Besnier, Isabelle; Talvinen, Kati; Dumez, Catherine; Vihola, Anna; Dubas, Frédéric; Fardeau, Michel; Hackman, Peter; Carpen, Olli; Udd, Bjarne

2006-07-01

Mutations in titin are well known cause of late onset autosomal dominant distal myopathy. Mutations in another sarcomeric protein, myotilin, were first identified in two families with dominant limb girdle muscular phenotype. Recently, however, myotilin mutations have been associated with more distal phenotypes in patients with late onset myofibrillar myopathy. We report here a multigenerational French family in which gene sequencing identified a S60F myotilin mutation in all patients with full penetrance despite very late onset. The family was originally reported as a distal myopathy but intrafamilial variability was remarkable with proximal or distal muscle weakness or both. Extended morphological characteristics of muscle biopsy findings in myotilinopathy indicate that immunohistochemistry may be important for selection of molecular genetic approach in myofibrillar myopathy.

Performance evaluation of the HepB Typer-Entecavir kit for detection of entecavir resistance mutations in chronic hepatitis B.

PubMed

Ahn, Sang Hoon; Chun, Ji-Yong; Shin, Soo-Kyung; Park, Jun Yong; Yoo, Wangdon; Hong, Sun Pyo; Kim, Soo-Ok; Han, Kwang-Hyub

2013-12-01

Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B.

A repetitive mutation and selection system for bacterial evolution to increase the specific affinity to pancreatic cancer cells.

PubMed

Osawa, Masaki

2018-01-01

It is difficult to target and kill cancer cells. One possible approach is to mutate bacteria to enhance their binding to cancer cells. In the present study, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were randomly mutated, and then were positively and negatively selected for binding cancer vs normal cells. With repetitive mutation and selection both bacteria successfully evolved to increase affinity to the pancreatic cancer cell line (Mia PaCa-2) but not normal cells (HPDE: immortalized human pancreatic ductal epithelial cells). The mutant E. coli and B. subtilis strains bound to Mia PaCa-2 cells about 10 and 25 times more than to HPDE cells. The selected E. coli strain had mutations in biofilm-related genes and the regulatory region for a type I pilus gene. Consistent with type I pili involvement, mannose could inhibit the binding to cells. The results suggest that weak but specific binding is involved in the initial step of adhesion. To test their ability to kill Mia PaCa-2 cells, hemolysin was expressed in the mutant strain. The hemolysin released from the mutant strain was active and could kill Mia PaCa-2 cells. In the case of B. subtilis, the initial binding to the cells was a weak interaction of the leading pole of the motile bacteria. The frequency of this interaction to Mia PaCa-2 cells dramatically increased in the evolved mutant strain. This mutant strain could also specifically invade beneath Mia PaCa-2 cells and settle there. This type of mutation/selection strategy may be applicable to other combinations of cancer cells and bacterial species.

Mild Myopathy Is Associated with COMP but Not MATN3 Mutations in Mouse Models of Genetic Skeletal Diseases

PubMed Central

Piróg, Katarzyna A.; Katakura, Yoshihisa; Mironov, Aleksandr; Briggs, Michael D.

2013-01-01

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are skeletal disorders resulting from mutations in COMP, matrilin-3 or collagen IX and are characterised by short-limbed dwarfism and premature osteoarthritis. Interestingly, recent reports suggest patients can also manifest with muscle weakness. Here we present a detailed analysis of two mouse models of the PSACH/MED disease spectrum; ΔD469 T3-COMP (PSACH) and V194D matrilin-3 (MED). In grip test experiments T3-COMP mice were weaker than wild-type littermates, whereas V194D mice behaved as controls, confirming that short-limbed dwarfism alone does not contribute to PSACH/MED-related muscle weakness. Muscles from T3-COMP mice showed an increase in centronuclear fibers at the myotendinous junction. T3-COMP tendons became more lax in cyclic testing and showed thicker collagen fibers when compared with wild-type tissue; matrilin-3 mutant tissues were indistinguishable from controls. This comprehensive study of the myopathy associated with PSACH/MED mutations enables a better understanding of the disease progression, confirms that it is genotype specific and that the limb weakness originates from muscle and tendon pathology rather than short-limbed dwarfism itself. Since some patients are primarily diagnosed with neuromuscular symptoms, this study will facilitate better awareness of the differential diagnoses that might be associated with the PSACH/MED spectrum and subsequent care of PSACH/MED patients. PMID:24312420

Assessment of Depression, Anxiety, Quality of Life, and Coping in Long-Standing Multiple Endocrine Neoplasia Type 2 Patients.

PubMed

Rodrigues, Karine C; Toledo, Rodrigo A; Coutinho, Flavia L; Nunes, Adriana B; Maciel, Rui M B; Hoff, Ana O; Tavares, Marcos C; Toledo, Sergio P A; Lourenço, Delmar M

2017-05-01

Data on psychological harm in multiple endocrine neoplasia type 2 (MEN2) are scarce. The aim of this study was to assess anxiety, depression, quality of life, and coping in long-standing MEN2 patients. Patients were 43 adults (age ≥18 years) with clinical and genetic diagnosis of MEN2 and long-term follow-up (10.6 ± 8.2 years; range 1-33 years). This was a cross-sectional study with qualitative and quantitative psychological assessment using semi-directed interviews and HADS, EORTC QLQ C30, and MINI-MAC scales. Adopting clinical criteria from 2015 ATA Guidelines on MEN2, biochemical cure (39%; 16/41), persistence/recurrence (61%; 25/41), and stable chronic disease (22/41) of medullary thyroid carcinoma (MTC) were scored. Pheochromocytoma affected 19 (44%) patients, with previous adrenalectomy in 17 of them. Overall, anxiety (42%; mean score 11 ± 2.9; range 8-18; anxiety is defined as a score ≥8) and depression (26%; mean score 11 ± 3.8; range 8-20; depression is defined as a score ≥8) symptoms were frequent. Patients who transmitted RET mutations to a child had higher scores for weakness-discouragement/anxious preoccupation and lower scores for cognitive, emotional, and physical functioning (p < 0.05). Feelings of guilt were present in 35% of patients with mutation-positive children. Lower mean score values for depression and anxiety and higher scores for role, cognitive, and emotional functioning were noticed in 33 patients who were well-informed about their disease (p < 0.05). Fighting spirit was more frequently found in patients with multiple surgical procedures (p = 0.019) and controlled chronic adrenal insufficiency (p = 0.024). Patients with MEN2-related stress-inducing factors had lower scores for fighting spirit and cognitive functioning and higher scores for insomnia and dyspnea (p < 0.05). Eleven patients required sustained psychotherapeutic treatment. Mean global health status was relatively good in MEN2 cases (68.1 ± 22.3), and the cured group had higher physical functioning (p = 0.021). Psychological distress is likely chronic in MEN2 patients. This study identified diverse MEN2-related factors (degree of information on disease, mutation-positive children, number of surgeries, comorbidities, stress-inducing factors, and cure) interfering positively or negatively with the results of the psychometrics scales. The active investigation of these factors and the applied psychological assessment protocol are useful to identify MEN2 patients requiring psychological assistance.

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations.

PubMed

Pfaller, Christian K; Mastorakos, George M; Matchett, William E; Ma, Xiao; Samuel, Charles E; Cattaneo, Roberto

2015-08-01

Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

PubMed Central

Pfaller, Christian K.; Mastorakos, George M.; Matchett, William E.; Ma, Xiao; Samuel, Charles E.

2015-01-01

ABSTRACT Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant. PMID:25972541

Aligning a Receiving Antenna Array to Reduce Interference

NASA Technical Reports Server (NTRS)

Jongeling, Andre P.; Rogstad, David H.

2009-01-01

A digital signal-processing algorithm has been devised as a means of aligning (as defined below) the outputs of multiple receiving radio antennas in a large array for the purpose of receiving a desired weak signal transmitted by a single distant source in the presence of an interfering signal that (1) originates at another source lying within the antenna beam and (2) occupies a frequency band significantly wider than that of the desired signal. In the original intended application of the algorithm, the desired weak signal is a spacecraft telemetry signal, the antennas are spacecraft-tracking antennas in NASA s Deep Space Network, and the source of the wide-band interfering signal is typically a radio galaxy or a planet that lies along or near the line of sight to the spacecraft. The algorithm could also afford the ability to discriminate between desired narrow-band and nearby undesired wide-band sources in related applications that include satellite and terrestrial radio communications and radio astronomy. The development of the present algorithm involved modification of a prior algorithm called SUMPLE and a predecessor called SIMPLE. SUMPLE was described in Algorithm for Aligning an Array of Receiving Radio Antennas (NPO-40574), NASA Tech Briefs Vol. 30, No. 4 (April 2006), page 54. To recapitulate: As used here, aligning signifies adjusting the delays and phases of the outputs from the various antennas so that their relatively weak replicas of the desired signal can be added coherently to increase the signal-to-noise ratio (SNR) for improved reception, as though one had a single larger antenna. Prior to the development of SUMPLE, it was common practice to effect alignment by means of a process that involves correlation of signals in pairs. SIMPLE is an example of an algorithm that effects such a process. SUMPLE also involves correlations, but the correlations are not performed in pairs. Instead, in a partly iterative process, each signal is appropriately weighted and then correlated with a composite signal equal to the sum of the other signals.

Increased T-cell receptor mutation frequency in radiation-exposed residents living near the Semipalatinsk nuclear test site.

PubMed

Taooka, Yasuyuki; Takeichi, Nobuo; Noso, Yoshihiro; Kawano, Noriyuki; Apsalikov, Kazbek N; Hoshi, Masaharu

2006-02-01

From 1949 to 1989, 488 nuclear explosions were carried out in Semipalatinsk, and the cancer risk is increased in this region. Measuring somatic-cell mutation frequencies may be a useful tool for evaluating cancer risk within radiation-exposed populations. Here, we report the first evidence of increased T-cell receptor (TCR) mutations in peripheral blood from radiation-exposed residents of Semipalatinsk. The TCR mutation frequency in the highly exposed residents (Dolon and Sarzhal) was significantly higher than in the control group (Kokpekti). There was no statistically significant difference between the control group and the weakly exposed group (Kaynar and Semipalatinsk-city). The TCR mutation assay appeared to be a useful biological dosimeter even after a period of 40 years since radiation exposure. This may be the result of specific conditions, such as the presence of internal exposure.

Mistranslation can enhance fitness through purging of deleterious mutations

PubMed Central

Bratulic, Sinisa; Toll-Riera, Macarena; Wagner, Andreas

2017-01-01

Phenotypic mutations are amino acid changes caused by mistranslation. How phenotypic mutations affect the adaptive evolution of new protein functions is unknown. Here we evolve the antibiotic resistance protein TEM-1 towards resistance on the antibiotic cefotaxime in an Escherichia coli strain with a high mistranslation rate. TEM-1 populations evolved in such strains endow host cells with a general growth advantage, not only on cefotaxime but also on several other antibiotics that ancestral TEM-1 had been unable to deactivate. High-throughput sequencing of TEM-1 populations shows that this advantage is associated with a lower incidence of weakly deleterious genotypic mutations. Our observations show that mistranslation is not just a source of noise that delays adaptive evolution. It could even facilitate adaptive evolution by exacerbating the effects of deleterious mutations and leading to their more efficient purging. The ubiquity of mistranslation and its effects render mistranslation an important factor in adaptive protein evolution. PMID:28524864

Sexual selection protects against extinction.

PubMed

Lumley, Alyson J; Michalczyk, Łukasz; Kitson, James J N; Spurgin, Lewis G; Morrison, Catriona A; Godwin, Joanne L; Dickinson, Matthew E; Martin, Oliver Y; Emerson, Brent C; Chapman, Tracey; Gage, Matthew J G

2015-06-25

Reproduction through sex carries substantial costs, mainly because only half of sexual adults produce offspring. It has been theorized that these costs could be countered if sex allows sexual selection to clear the universal fitness constraint of mutation load. Under sexual selection, competition between (usually) males and mate choice by (usually) females create important intraspecific filters for reproductive success, so that only a subset of males gains paternity. If reproductive success under sexual selection is dependent on individual condition, which is contingent to mutation load, then sexually selected filtering through 'genic capture' could offset the costs of sex because it provides genetic benefits to populations. Here we test this theory experimentally by comparing whether populations with histories of strong versus weak sexual selection purge mutation load and resist extinction differently. After evolving replicate populations of the flour beetle Tribolium castaneum for 6 to 7 years under conditions that differed solely in the strengths of sexual selection, we revealed mutation load using inbreeding. Lineages from populations that had previously experienced strong sexual selection were resilient to extinction and maintained fitness under inbreeding, with some families continuing to survive after 20 generations of sib × sib mating. By contrast, lineages derived from populations that experienced weak or non-existent sexual selection showed rapid fitness declines under inbreeding, and all were extinct after generation 10. Multiple mutations across the genome with individually small effects can be difficult to clear, yet sum to a significant fitness load; our findings reveal that sexual selection reduces this load, improving population viability in the face of genetic stress.

Mutations in PTRH2 cause novel infantile-onset multisystem disease with intellectual disability, microcephaly, progressive ataxia, and muscle weakness.

PubMed

Hu, Hao; Matter, Michelle L; Issa-Jahns, Lina; Jijiwa, Mayumi; Kraemer, Nadine; Musante, Luciana; de la Vega, Michelle; Ninnemann, Olaf; Schindler, Detlev; Damatova, Natalia; Eirich, Katharina; Sifringer, Marco; Schrötter, Sandra; Eickholt, Britta J; van den Heuvel, Lambert; Casamina, Chanel; Stoltenburg-Didinger, Gisela; Ropers, Hans-Hilger; Wienker, Thomas F; Hübner, Christoph; Kaindl, Angela M

2014-12-01

To identify the cause of a so-far unreported phenotype of infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD). We characterized a consanguineous family of Yazidian-Turkish descent with IMNEPD. The two affected children suffer from intellectual disability, postnatal microcephaly, growth retardation, progressive ataxia, distal muscle weakness, peripheral demyelinating sensorimotor neuropathy, sensorineural deafness, exocrine pancreas insufficiency, hypothyroidism, and show signs of liver fibrosis. We performed whole-exome sequencing followed by bioinformatic analysis and Sanger sequencing on affected and unaffected family members. The effect of mutations in the candidate gene was studied in wild-type and mutant mice and in patient and control fibroblasts. In a consanguineous family with two individuals with IMNEPD, we identified a homozygous frameshift mutation in the previously not disease-associated peptidyl-tRNA hydrolase 2 (PTRH2) gene. PTRH2 encodes a primarily mitochondrial protein involved in integrin-mediated cell survival and apoptosis signaling. We show that PTRH2 is highly expressed in the developing brain and is a key determinant in maintaining cell survival during human tissue development. Moreover, we link PTRH2 to the mTOR pathway and thus the control of cell size. The pathology suggested by the human phenotype and neuroimaging studies is supported by analysis of mutant mice and patient fibroblasts. We report a novel disease phenotype, show that the genetic cause is a homozygous mutation in the PTRH2 gene, and demonstrate functional effects in mouse and human tissues. Mutations in PTRH2 should be considered in patients with undiagnosed multisystem neurologic, endocrine, and pancreatic disease.

An mtDNA mutation accelerates liver aging by interfering with the ROS response and mitochondrial life cycle.

PubMed

Niemann, Jan; Johne, Cindy; Schröder, Susanne; Koch, Franziska; Ibrahim, Saleh M; Schultz, Julia; Tiedge, Markus; Baltrusch, Simone

2017-01-01

Mitochondrial dysfunction affects liver metabolism, but it remains unclear whether this interferes with normal liver aging. We investigated several mitochondrial pathways in hepatocytes and liver tissue from a conplastic mouse strain compared with the control C57BL/6NTac strain over 18 months of life. The C57BL/6NTac-mtNODLtJ mice differed from C57BL/6NTac mice by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase. Young C57BL/6NTac-mtNODLtJ mice showed reduced mitochondrial metabolism but similar reactive oxygen species (ROS) production to C57BL/6NTac mice. Whereas ROS increased almost equally up to 9 months in both strains, different mitochondrial adaptation strategies resulted in decreasing ROS in advanced age in C57BL/6NTac mice, but persistent ROS production in C57BL/6NTac-mtNODLtJ mice. Only the conplastic strain developed elongated mitochondrial networks with artificial loop structures, depressed autophagy, high mitochondrial respiration and up-regulated antioxidative response. Our results indicate that mtDNA mutations accelerate liver ballooning degeneration and carry a serious risk of premature organ aging. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of energetically biased transcripts of viruses and transposable elements

PubMed Central

Secolin, Rodrigo; Pascoal, Vinícius D’Ávila Bitencourt; Lopes-Cendes, Iscia; Pereira, Tiago Campos

2012-01-01

RNA interference (RNAi) is a natural endogenous process by which double-stranded RNA molecules trigger potent and specific gene silencing in eukaryotic cells and is characterized by target RNA cleavage. In mammals, small interfering RNAs (siRNAs) are the trigger molecules of choice and constitute a new class of RNA-based antiviral agents. In an efficient RNAi response, the antisense strand of siRNAs must enter the RNA-induced silencing complex (RISC) in a process mediated by thermodynamic features. In this report, we hypothesize that silent mutations capable of inverting thermodynamic properties can promote resistance to siRNAs. Extensive computational analyses were used to assess whether continuous selective pressure that promotes such mutations could lead to the emergence of viral strains completely resistant to RNAi (i.e., prone to transfer only the sense strands to RISC). Based on our findings, we propose that, although synonymous mutations may produce functional resistance, this strategy cannot be systematically adopted by viruses since the longest RNAi-refractory sequence is only 10 nt long. This finding also suggests that all mRNAs display fluctuating thermodynamic landscapes and that, in terms of thermodynamic features, RNAi is a very efficient antiviral system since there will always be sites susceptible to siRNAs. PMID:23271949

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

PubMed

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

2017-11-23

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

Primary adenosine monophosphate (AMP) deaminase deficiency in a hypotonic infant.

PubMed

Castro-Gago, Manuel; Gómez-Lado, Carmen; Pérez-Gay, Laura; Eirís-Puñal, Jesús; Martínez, Elena Pintos; García-Consuegra, Inés; Martín, Miguel Angel

2011-06-01

The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil.

PubMed

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Watanabe, Aripuanã Sakurada Aranha; Biselli-Périco, Joice Matos; Drumond, Betânia Paiva; Ribeiro, Milene Rocha; Vedovello, Danila; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; Chaves, Bárbara Aparecida; Silva, Mayara Marques Carneiro da; Batista, Izabella Cristina Andrade; Papa, Michelle Premazzi; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; Arruda, Luciana Barros de; Souza, Danielle da Glória de; Teixeira, Mauro Martins; Vasilakis, Nikos; Nogueira, Maurício Lacerda

2018-05-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil

PubMed Central

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Biselli-Périco, Joice Matos; Ribeiro, Milene Rocha; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; da Silva, Mayara Marques Carneiro; Batista, Izabella Cristina Andrade; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; de Souza, Danielle da Glória; Teixeira, Mauro Martins; Vasilakis, Nikos

2018-01-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto. PMID:29813061

Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype–Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy

PubMed Central

Böhm, Johann; Biancalana, Valérie; DeChene, Elizabeth T.; Bitoun, Marc; Pierson, Christopher R.; Schaefer, Elise; Karasoy, Hatice; Dempsey, Melissa A.; Klein, Fabrice; Dondaine, Nicolas; Kretz, Christine; Haumesser, Nicolas; Poirson, Claire; Toussaint, Anne; Greenleaf, Rebecca S.; Barger, Melissa A.; Mahoney, Lane J.; Kang, Peter B.; Zanoteli, Edmar; Vissing, John; Witting, Nanna; Echaniz-Laguna, Andoni; Wallgren-Pettersson, Carina; Dowling, James; Merlini, Luciano; Oldfors, Anders; Ousager, Lilian Bomme; Melki, Judith; Krause, Amanda; Jern, Christina; Oliveira, Acary S. B.; Petit, Florence; Jacquette, Aurélia; Chaussenot, Annabelle; Mowat, David; Leheup, Bruno; Cristofano, Michele; Aldea, Juan José Poza; Michel, Fabrice; Furby, Alain; Llona, Jose E. Barcena; Van Coster, Rudy; Bertini, Enrico; Urtizberea, Jon Andoni; Drouin-Garraud, Valérie; Béroud, Christophe; Prudhon, Bernard; Bedford, Melanie; Mathews, Katherine; Erby, Lori A. H.; Smith, Stephen A.; Roggenbuck, Jennifer; Crowe, Carol A.; Spitale, Allison Brennan; Johal, Sheila C.; Amato, Anthony A.; Demmer, Laurie A.; Jonas, Jessica; Darras, Basil T.; Bird, Thomas D.; Laurino, Mercy; Welt, Selman I.; Trotter, Cynthia; Guicheney, Pascale; Das, Soma; Mandel, Jean-Louis; Beggs, Alan H.; Laporte, Jocelyn

2012-01-01

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype–phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot–Marie–Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT. PMID:22396310

FAT1 Gene Alteration in Facioscapulohumeral Muscular Dystrophy Type 1.

PubMed

Park, Hyung Jun; Lee, Wookjae; Kim, Se Hoon; Lee, Jung Hwan; Shin, Ha Young; Kim, Seung Min; Park, Kee Duk; Lee, Ji Hyun; Choi, Young Chul

2018-03-01

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array. Recent studies revealed that the FAT1 expression is associated with disease activity of FSHD, and the FAT1 alterations result in myopathy with a FSHD-like phenotype. We describe a 59-year-old woman with both contracted D4Z4 repeat units and a FAT1 mutation. Shoulder girdle muscle weakness developed at the age of 56 years, and was followed by proximal leg weakness. When we examined her at 59 years of age, she displayed asymmetric and predominant weakness of facial and proximal muscles. Muscle biopsy showed increased variation in fiber size and multifocal degenerating fibers with lymphocytic infiltration. Southern blot analysis revealed 8 D4Z4 repeat units, and targeted sequencing of modifier genes demonstrated the c.10331 A>G variant in the FAT1 gene. This FAT1 variant has previously been reported as pathogenic variant in a patient with FSHD-like phenotype. Our study is the first report of a FAT1 mutation in a FSHD1 patient, and suggests that FAT1 alterations might work as a genetic modifier. © Copyright: Yonsei University College of Medicine 2018.

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

PubMed Central

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

1994-01-01

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

Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses

PubMed Central

Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi

2016-01-01

ABSTRACT HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro. This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals. PMID:27903797

Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses.

PubMed

Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2017-02-15

HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals. Copyright © 2017 American Society for Microbiology.

Performance evaluation of the HepB Typer-Entecavir kit for detection of entecavir resistance mutations in chronic hepatitis B

PubMed Central

Ahn, Sang Hoon; Chun, Ji-Yong; Shin, Soo-Kyung; Park, Jun Yong; Yoo, Wangdon; Hong, Sun Pyo; Han, Kwang-Hyub

2013-01-01

Background/Aims Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. Methods The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. Results Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. Conclusions The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B. PMID:24459645

Parent-progeny sequencing indicates higher mutation rates in heterozygotes.

PubMed

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

2015-07-23

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

NIPA1 Gene Mutations Cause Autosomal Dominant Hereditary Spastic Paraplegia (SPG6)

PubMed Central

Rainier, Shirley; Chai, Jing-Hua; Tokarz, Debra; Nicholls, Robert D.; Fink, John K.

2003-01-01

The hereditary spastic paraplegias (HSPs) are genetically heterogeneous disorders characterized by progressive lower-extremity weakness and spasticity. The molecular pathogenesis is poorly understood. We report discovery of a dominant negative mutation in the NIPA1 gene in a kindred with autosomal dominant HSP (ADHSP), linked to chromosome 15q11-q13 (SPG6 locus); and precisely the same mutation in an unrelated kindred with ADHSP that was too small for meaningful linkage analysis. NIPA1 is highly expressed in neuronal tissues and encodes a putative membrane transporter or receptor. Identification of the NIPA1 function and ligand will aid an understanding of axonal neurodegeneration in HSP and may have important therapeutic implications. PMID:14508710

Leigh syndrome with Fukuyama congenital muscular dystrophy: a case report.

PubMed

Kondo, Hidehito; Tanda, Koichi; Tabata, Chihiro; Hayashi, Kohei; Kihara, Minako; Kizaki, Zenro; Taniguchi-Ikeda, Mariko; Mori, Masato; Murayama, Kei; Ohtake, Akira

2014-09-01

We report the first case of Leigh syndrome (LS) with Fukuyama congenital muscular dystrophy (FCMD). A neonate suffered from lactic acidosis and subsequently presented with poor feeding, muscle weakness, hypotonia, cardiopulmonary dysfunction, and hydrocephalus. He died at 17 months. The findings of brain magnetic resonance imaging indicated some specific features of both LS and FCMD, and FCMD gene mutation was detected. Decreased mitochondrial respiratory complex I and II activity was noted. Mitochondrial DNA sequencing showed no pathogenic mutation. A case with complex I+II deficiency has rarely been reported, suggesting a nuclear gene mutation. Copyright © 2013 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients.

PubMed

Cabagnols, Xénia; Favale, Fabrizia; Pasquier, Florence; Messaoudi, Kahia; Defour, Jean Philippe; Ianotto, Jean Christophe; Marzac, Christophe; Le Couédic, Jean Pierre; Droin, Nathalie; Chachoua, Ilyas; Favier, Remi; Diop, M'boyba Khadija; Ugo, Valérie; Casadevall, Nicole; Debili, Najet; Raslova, Hana; Bellanné-Chantelot, Christine; Constantinescu, Stefan N; Bluteau, Olivier; Plo, Isabelle; Vainchenker, William

2016-01-21

Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN. © 2016 by The American Society of Hematology.

Mutations in PTRH2 cause novel infantile-onset multisystem disease with intellectual disability, microcephaly, progressive ataxia, and muscle weakness

PubMed Central

Hu, Hao; Matter, Michelle L; Issa-Jahns, Lina; Jijiwa, Mayumi; Kraemer, Nadine; Musante, Luciana; de la Vega, Michelle; Ninnemann, Olaf; Schindler, Detlev; Damatova, Natalia; Eirich, Katharina; Sifringer, Marco; Schrötter, Sandra; Eickholt, Britta J; van den Heuvel, Lambert; Casamina, Chanel; Stoltenburg-Didinger, Gisela; Ropers, Hans-Hilger; Wienker, Thomas F; Hübner, Christoph; Kaindl, Angela M

2014-01-01

Objective To identify the cause of a so-far unreported phenotype of infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD). Methods We characterized a consanguineous family of Yazidian-Turkish descent with IMNEPD. The two affected children suffer from intellectual disability, postnatal microcephaly, growth retardation, progressive ataxia, distal muscle weakness, peripheral demyelinating sensorimotor neuropathy, sensorineural deafness, exocrine pancreas insufficiency, hypothyroidism, and show signs of liver fibrosis. We performed whole-exome sequencing followed by bioinformatic analysis and Sanger sequencing on affected and unaffected family members. The effect of mutations in the candidate gene was studied in wild-type and mutant mice and in patient and control fibroblasts. Results In a consanguineous family with two individuals with IMNEPD, we identified a homozygous frameshift mutation in the previously not disease-associated peptidyl-tRNA hydrolase 2 (PTRH2) gene. PTRH2 encodes a primarily mitochondrial protein involved in integrin-mediated cell survival and apoptosis signaling. We show that PTRH2 is highly expressed in the developing brain and is a key determinant in maintaining cell survival during human tissue development. Moreover, we link PTRH2 to the mTOR pathway and thus the control of cell size. The pathology suggested by the human phenotype and neuroimaging studies is supported by analysis of mutant mice and patient fibroblasts. Interpretation We report a novel disease phenotype, show that the genetic cause is a homozygous mutation in the PTRH2 gene, and demonstrate functional effects in mouse and human tissues. Mutations in PTRH2 should be considered in patients with undiagnosed multisystem neurologic, endocrine, and pancreatic disease. PMID:25574476

A novel mouse model carrying a human cytoplasmic dynein mutation shows motor behavior deficits consistent with Charcot-Marie-Tooth type 2O disease.

PubMed

Sabblah, Thywill T; Nandini, Swaran; Ledray, Aaron P; Pasos, Julio; Calderon, Jami L Conley; Love, Rachal; King, Linda E; King, Stephen J

2018-01-29

Charcot-Marie-Tooth disease (CMT) is a peripheral neuromuscular disorder in which axonal degeneration causes progressive loss of motor and sensory nerve function. The loss of motor nerve function leads to distal muscle weakness and atrophy, resulting in gait problems and difficulties with walking, running, and balance. A mutation in the cytoplasmic dynein heavy chain (DHC) gene was discovered to cause an autosomal dominant form of the disease designated Charcot-Marie-Tooth type 2 O disease (CMT2O) in 2011. The mutation is a single amino acid change of histidine into arginine at amino acid 306 (H306R) in DHC. In order to understand the onset and progression of CMT2, we generated a knock-in mouse carrying the corresponding CMT2O mutation (H304R/+). We examined H304R/+ mouse cohorts in a 12-month longitudinal study of grip strength, tail suspension, and rotarod assays. H304R/+ mice displayed distal muscle weakness and loss of motor coordination phenotypes consistent with those of individuals with CMT2. Analysis of the gastrocnemius of H304R/+ male mice showed prominent defects in neuromuscular junction (NMJ) morphology including reduced size, branching, and complexity. Based on these results, the H304R/+ mouse will be an important model for uncovering functions of dynein in complex organisms, especially related to CMT onset and progression.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-12-10

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

Cationic lipid-assisted polymeric nanoparticle mediated GATA2 siRNA delivery for synthetic lethal therapy of KRAS mutant non-small-cell lung carcinoma.

PubMed

Shen, Song; Mao, Chong-Qiong; Yang, Xian-Zhu; Du, Xiao-Jiao; Liu, Yang; Zhu, Yan-Hua; Wang, Jun

2014-08-04

Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy.

Epidemiology of echinocandin resistance in Candida.

PubMed

Grossman, Nina T; Chiller, Tom M; Lockhart, Shawn R

2014-12-01

Echinocandins are the newest antifungal agents approved for use in treating Candida infections in the US. They act by interfering with 1,3-β-D-glucan synthase and therefore disrupt cell wall production and lead to Candida cell death. There is no intrinsic resistance to echinocandins among Candida species, and isolates from historic collections archived before the release of the echinocandins show no resistance. Resistance to the echinocandins remains low among most Candida species and ranges overall from 0-1%. Among isolates of Candida glabrata , the proportion of resistant isolates is higher and has been reported to be as high as 13.5% in at least one hospital. Antifungal resistance is due to specific amino acid mutations in the Fksp subunit(s) of the 1,3-β-D-glucan synthase protein which are localized to one of two hotspots. These mutations are being recognized in isolates from patients who have failed echinocandin therapy, and often lead to a poor outcome. While the future looks bright for the echinocandins against most Candida species, C. glabrata remains a species of concern and resistance rates of C. glabrata to the echinocandins should be monitored closely.

KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: A meta-analysis of 41 studies

PubMed Central

Pan, Wei; Yang, Yan; Zhu, Hongcheng; Zhang, Youcheng; Zhou, Rongping; Sun, Xinchen

2016-01-01

Mutation of oncogene KRAS is common in non-small cell lung cancer (NSCLC), however, its clinical significance is still controversial. Independent studies evaluating its prognostic and predictive value usually drew inconsistent conclusions. Hence, We performed a meta-analysis with 41 relative publications, retrieved from multi-databases, to reconcile these controversial results and to give an overall impression of KRAS mutation in NSCLC. According to our findings, KRAS mutation was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in early stage resected NSCLC (hazard ratio or HR=1.56 and 1.57, 95% CI 1.39-1.76 and 1.17-2.09 respectively), and with inferior outcomes of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) treatment and chemotherapy (relative risk or RR=0.21 and 0.66 for objective response rate or ORR, 95% CI 0.12-0.39 and 0.54-0.81 respectively; HR=1.46 and 1.30 for progression-free survival or PFS, 95%CI 1.23-1.74 and 1.14-1.50 respectively) in advanced NSCLC. When EGFR mutant patients were excluded, KRAS mutation was still significantly associated with worse OS and PFS of EGFR-TKIs (HR=1.40 and 1.35, 95 % CI 1.21-1.61 and 1.11-1.64). Although KRAS mutant patients presented worse DFS and PFS of chemotherapy (HR=1.33 and 1.11, 95% CI 0.97-1.84 and 0.95-1.30), and lower response rate to EGFR-TKIs or chemotherapy (RR=0.55 and 0.88, 95 % CI 0.27-1.11 and 0.76-1.02), statistical differences were not met. In conclusion, KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC. There's a need for developing target therapies for KRAS mutant lung cancer and other tumors. PMID:26840022

Bm-muted, orthologous to mouse muted and encoding a subunit of the BLOC-1 complex, is responsible for the otm translucent mutation of the silkworm Bombyx mori.

PubMed

Zhang, Haokun; Kiuchi, Takashi; Wang, Lingyan; Kawamoto, Munetaka; Suzuki, Yutaka; Sugano, Sumio; Banno, Yutaka; Katsuma, Susumu; Shimada, Toru

2017-09-20

"Tanaka's mottled translucent" (otm) is a mutation of the silkworm Bombyx mori that exhibits translucent skin during larval stages. We performed positional cloning of the gene responsible for otm and mapped it to a 364-kb region on chromosome 5 that contains 22 hypothetical protein-coding genes. We performed RNA-seq analysis of the epidermis and fat body of otm larvae and determined that the gene BGIBMGA002619 may be responsible for the otm mutation. BGIBMGA002619 encodes the biosynthesis of lysosome-related organelles complex 1 (BLOC-1) subunit 5, whose ortholog is responsible for the Muted mutant in mouse. Accordingly, we named this gene Bm-muted. We discovered that the expression of Bm-muted in the epidermis and fat body of otm mutants was dramatically suppressed compared with the wild type. We determined the nucleotide sequences of the full-length cDNA and genomic region corresponding to Bm-muted and found that a 538-bp long DNA sequence similar to B. mori transposon Organdy was inserted into the 3' end of the first intron of Bm-muted in two otm strains. The Bm-muted cDNA of otm mutants lacked exon 2, and accordingly generated a premature stop codon in exon 3. In addition, short interfering RNA (siRNA)-mediated knockdown of this gene caused localized partial translucency of larval skin. These data indicate that the mutation in Bm-muted caused the otm-mutant phenotype. We propose that the insertion of Organdy caused a splicing disorder in Bm-muted in the otm mutant, resulting in a null mutation of Bm-muted. This mutation is likely to cause deficiencies in urate granule formation in epidermal cells that result in translucent larval skin. Copyright © 2017 Elsevier B.V. All rights reserved.

Emergence of the virulence-associated PB2 E627K substitution in a fatal human case of highly pathogenic avian influenza virus A(H7N7) infection as determined by Illumina ultra-deep sequencing.

PubMed

Jonges, Marcel; Welkers, Matthijs R A; Jeeninga, Rienk E; Meijer, Adam; Schneeberger, Peter; Fouchier, Ron A M; de Jong, Menno D; Koopmans, Marion

2014-02-01

Avian influenza viruses are capable of crossing the species barrier and infecting humans. Although evidence of human-to-human transmission of avian influenza viruses to date is limited, evolution of variants toward more-efficient human-to-human transmission could result in a new influenza virus pandemic. In both the avian influenza A(H5N1) and the recently emerging avian influenza A(H7N9) viruses, the polymerase basic 2 protein (PB2) E627K mutation appears to be of key importance for human adaptation. During a large influenza A(H7N7) virus outbreak in the Netherlands in 2003, the A(H7N7) virus isolated from a fatal human case contained the PB2 E627K mutation as well as a hemagglutinin (HA) K416R mutation. In this study, we aimed to investigate whether these mutations occurred in the avian or the human host by Illumina Ultra-Deep sequencing of three previously uninvestigated clinical samples obtained from the fatal case. In addition, we investigated three chicken samples, two of which were obtained from the source farm. Results showed that the PB2 E627K mutation was not present in any of the chicken samples tested. Surprisingly, the avian samples were characterized by the presence of influenza virus defective RNA segments, suggestive for the synthesis of defective interfering viruses during infection in poultry. In the human samples, the PB2 E627K mutation was identified with increasing frequency during infection. Our results strongly suggest that human adaptation marker PB2 E627K has emerged during virus infection of a single human host, emphasizing the importance of reducing human exposure to avian influenza viruses to reduce the likelihood of viral adaptation to humans.

Long-term outcome of Leigh syndrome caused by the NARP-T8993C mtDNA mutation.

PubMed

Debray, François-Guillaume; Lambert, Marie; Lortie, Anne; Vanasse, Michel; Mitchell, Grant A

2007-09-01

Mutations at mitochondrial DNA (mtDNA) nucleotide 8993 can cause neurogenic weakness, ataxia and retinitis pigmentosa (NARP syndrome), or maternally inherited Leigh syndrome (LS), with a correlation between the amount of mutant mtDNA and the severity of the neurological disease. The T8993C mutation is generally considered to be clinically milder than the T8993G mutation but when the level of heteroplasmy exceeds 90%, progressive neurodegeneration has been found. We report on a long-term follow-up of a patient who presented at 4 years of age with typical LS but showed an unexpected resolution of his symptoms and a favorable outcome. At 18 years of age, his neurological examination was near normal, with neither peripheral neuropathy nor retinopathy. mtDNA analysis identified the presence of T8993C mutation at high level (>95%) in the patient's blood leukocytes. This case report and literature review emphasizes the variability of the phenotypic expression of the T8993C mutation and the need for caution in predictive counseling in such patients. (c) 2007 Wiley-Liss, Inc. Copyright 2007 Wiley-Liss, Inc.

In silico Analysis of Conformational Changes Induced by Mutation of Aromatic Binding Residues: Consequences for Drug Binding in the hERG K+ Channel

PubMed Central

Knape, Kirsten; Linder, Tobias; Wolschann, Peter; Beyer, Anton; Stary-Weinzinger, Anna

2011-01-01

Pharmacological inhibition of cardiac hERG K+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences. PMID:22194911

Evolution of high-level resistance during low-level antibiotic exposure.

PubMed

Wistrand-Yuen, Erik; Knopp, Michael; Hjort, Karin; Koskiniemi, Sanna; Berg, Otto G; Andersson, Dan I

2018-04-23

It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both?

PubMed

Nissan, Moriah H; Solit, David B

2011-12-01

Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

PubMed Central

2018-01-01

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutations increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed. PMID:29442996

Quantum interference control of an isolated resonance lifetime in the weak-field limit.

PubMed

García-Vela, A

2015-11-21

Resonance states play an important role in a large variety of physical and chemical processes. Thus, controlling the resonance behavior, and particularly a key property like the resonance lifetime, opens up the possibility of controlling those resonance mediated processes. While such a resonance control is possible by applying strong-field approaches, the development of flexible weak-field control schemes that do not alter significantly the system dynamics still remains a challenge. In this work, one such control scheme within the weak-field regime is proposed for the first time in order to modify the lifetime of an isolated resonance state. The basis of the scheme suggested is quantum interference between two pathways induced by laser fields, that pump wave packet amplitude to the target resonance under control. The simulations reported here show that the scheme allows for both enhancement and quenching of the resonance survival lifetime, being particularly flexible to achieve large lifetime enhancements. Control effects on the resonance lifetime take place only while the pulse is operating. In addition, the conditions required to generate the two interfering quantum pathways are found to be rather easy to meet for general systems, which makes the experimental implementation straightforward and implies the wide applicability of the control scheme.

The investigation of genetic and clinical features in Chinese patients with juvenile amyotrophic lateral sclerosis.

PubMed

Liu, Z-J; Lin, H-X; Liu, G-L; Tao, Q-Q; Ni, W; Xiao, B-G; Wu, Z-Y

2017-09-01

Juvenile amyotrophic lateral sclerosis (JALS) occurs at an age of onset below 25 years with a heterogeneous disease onset location, variable progression and survival time. To investigate whether an ALS gene profile could resolve any aspects of clinical symptom heterogeneity, we have used targeted sequencing technology in a cohort of 12 JALS patients of Chinese descent. We detected 5 likely pathogenic mutations, 2 in familial probands and 3 in sporadic patients. One was a known TARDBP mutation (p.G348V) and 4 were FUS frameshift mutations including a known p.Gln519Ilefs*9 mutation and 3 novel mutations, p.Gly515Valfs*14, p.Gly486Profs*30, and p.Arg498Alafs*32. Of the 4 FUS mutations, 2 were able to be confirmed as de novo mutations. The TARDBP mutation carrier showed a classic ALS phenotype. All patients with FUS mutations experienced limb weakness at an early age and developed bulbar symptoms during the disease course. FUS mutations have previously been associated with increased JALS disease progression, however, we found a large range 12 to 84 months in disease survival (mean 58.2 months). Our results justify future screening for variants in FUS as it remains the most frequent genetic determinant of early onset, JALS (found in 30% of our patients). © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Swarm satellite mission scheduling & planning using Hybrid Dynamic Mutation Genetic Algorithm

NASA Astrophysics Data System (ADS)

Zheng, Zixuan; Guo, Jian; Gill, Eberhard

2017-08-01

Space missions have traditionally been controlled by operators from a mission control center. Given the increasing number of satellites for some space missions, generating a command list for multiple satellites can be time-consuming and inefficient. Developing multi-satellite, onboard mission scheduling & planning techniques is, therefore, a key research field for future space mission operations. In this paper, an improved Genetic Algorithm (GA) using a new mutation strategy is proposed as a mission scheduling algorithm. This new mutation strategy, called Hybrid Dynamic Mutation (HDM), combines the advantages of both dynamic mutation strategy and adaptive mutation strategy, overcoming weaknesses such as early convergence and long computing time, which helps standard GA to be more efficient and accurate in dealing with complex missions. HDM-GA shows excellent performance in solving both unconstrained and constrained test functions. The experiments of using HDM-GA to simulate a multi-satellite, mission scheduling problem demonstrates that both the computation time and success rate mission requirements can be met. The results of a comparative test between HDM-GA and three other mutation strategies also show that HDM has outstanding performance in terms of speed and reliability.

A new case of limb girdle muscular dystrophy 2G in a Greek patient, founder effect and review of the literature.

PubMed

Brusa, Roberta; Magri, Francesca; Papadimitriou, Dimitra; Govoni, Alessandra; Del Bo, Roberto; Ciscato, Patrizia; Savarese, Marco; Cinnante, Claudia; Walter, Maggie C; Abicht, Angela; Bulst, Stefanie; Corti, Stefania; Moggio, Maurizio; Bresolin, Nereo; Nigro, Vincenzo; Comi, Giacomo Pietro

2018-04-13

Limb girdle muscular dystrophy (LGMD) type 2G is a rare form of muscle disease, described only in a few patients worldwide, caused by mutations in TCAP gene, encoding the protein telethonin. It is characterised by proximal limb muscle weakness associated with distal involvement of lower limbs, starting in the first or second decade of life. We describe the case of a 37-year-old woman of Greek origin, affected by disto-proximal lower limb weakness. No cardiac or respiratory involvement was detected. Muscle biopsy showed myopathic changes with type I fibre hypotrophy, cytoplasmic vacuoles, lipid overload, multiple central nuclei and fibre splittings; ultrastructural examination showed metabolic abnormalities. Next generation sequencing analysis detected a homozygous frameshift mutation in the TCAP gene (c.90_91del), previously described in one Turkish family. Immunostaining and Western blot analysis showed complete absence of telethonin. Interestingly, Single Nucleotide Polymorphism analysis of the 10 Mb genomic region containing the TCAP gene showed a shared homozygous haplotype of both the Greek and the Turkish patients, thus suggesting a possible founder effect of TCAP gene c.90_91del mutation in this part of the Mediterranean area. Copyright © 2018 Elsevier B.V. All rights reserved.

Antisense oligonucleotide therapeutics for iron-sulphur cluster deficiency myopathy.

PubMed

Kollberg, Gittan; Holme, Elisabeth

2009-12-01

Iron-sulphur cluster deficiency myopathy is caused by a deep intronic mutation in ISCU resulting in inclusion of a cryptic exon in the mature mRNA. ISCU encodes the iron-sulphur cluster assembly protein IscU. Iron-sulphur clusters are essential for most basic redox transformations including the respiratory-chain function. Most patients are homozygous for the mutation with a phenotype characterized by a non-progressive myopathy with childhood onset of early fatigue, dyspnoea and palpitation on trivial exercise. A more severe phenotype with early onset of a slowly progressive severe muscle weakness, severe exercise intolerance and cardiomyopathy is caused by a missense mutation in compound with the intronic mutation. Treatment of cultured fibroblasts derived from three homozygous patients with an antisense phosphorodiamidate morpholino oligonucleotide for 48 h resulted in 100% restoration of the normal splicing pattern. The restoration was stable and after 21 days the correctly spliced mRNA still was the dominating RNA species.

[The role of remodeling complexes CHD1 and ISWI in spontaneous and UV-induced mutagenesis control in yeast Saccharomyces cerevisiae].

PubMed

Evstiukhina, T A; Alekseeva, E A; Fedorov, D V; Peshekhonov, V T; Korolev, V G

2017-02-01

Chromatin remodulators are special multiprotein machines capable of transforming the structure, constitution, and positioning of nucleosomes on DNA. Biochemical activities of remodeling complexes CHD1 and ISWI from the SWI2/SNF2 family are well established. They ensure correct positioning of nucleosomes along the genome, which is probably critical for genome stability, in particular, after action of polymerases, repair enzymes, and transcription. In this paper, we show that single mutations in genes ISW1, ISW2, and CHD1 weakly affect repair and mutagenic processes in yeast cells. At the same time, there are differences in the effect of these mutations on spontaneous mutation levels, which indicates certain specificity of action of protein complexes ISW1, ISW2, and CHD1 on expression of different genes that control repair and mutation processes in yeast.

Phenotype variability of infantile-onset multisystem neurologic, endocrine, and pancreatic disease IMNEPD.

PubMed

Picker-Minh, Sylvie; Mignot, Cyril; Doummar, Diane; Hashem, Mais; Faqeih, Eissa; Josset, Patrice; Dubern, Béatrice; Alkuraya, Fowzan S; Kraemer, Nadine; Kaindl, Angela M

2016-04-29

Infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD) has been recently linked to biallelic mutation of the peptidyl-tRNA hydrolase 2 gene PTRH2. Two index patients with IMNEPD in the original report had multiple neurological symptoms such as postnatal microcephaly, intellectual disability, developmental delay, sensorineural deafness, cerebellar atrophy, ataxia, and peripheral neuropathy. In addition, distal muscle weakness and abnormalities of thyroid, pancreas, and liver were found. Here, we report five further IMNEPD patients with a different homozygous PTRH2 mutation, broaden the phenotypic spectrum of the disease and differentiate common symptoms and interindividual variability in IMNEPD associated with a unique mutation. We thereby hope to better define IMNEPD and promote recognition and diagnosis of this novel disease entity.

Emerging understanding of the genotype-phenotype relationship in amyotrophic lateral sclerosis.

PubMed

Goutman, Stephen A; Chen, Kevin S; Paez-Colasante, Ximena; Feldman, Eva L

2018-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive, noncurable neurodegenerative disorder of the upper and lower motor neurons causing weakness and death within a few years of symptom onset. About 10% of patients with ALS have a family history of the disease; however, ALS-associated genetic mutations are also found in sporadic cases. There are over 100 ALS-associated mutations, and importantly, several genetic mutations, including C9ORF72, SOD1, and TARDBP, have led to mechanistic insight into this complex disease. In the clinical realm, knowledge of ALS genetics can also help explain phenotypic heterogeneity, aid in genetic counseling, and in the future may help direct treatment efforts. Copyright © 2018 Elsevier B.V. All rights reserved.

The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis.

PubMed

Imamura, Keiko; Izumi, Yuishin; Watanabe, Akira; Tsukita, Kayoko; Woltjen, Knut; Yamamoto, Takuya; Hotta, Akitsu; Kondo, Takayuki; Kitaoka, Shiho; Ohta, Akira; Tanaka, Akito; Watanabe, Dai; Morita, Mitsuya; Takuma, Hiroshi; Tamaoka, Akira; Kunath, Tilo; Wray, Selina; Furuya, Hirokazu; Era, Takumi; Makioka, Kouki; Okamoto, Koichi; Fujisawa, Takao; Nishitoh, Hideki; Homma, Kengo; Ichijo, Hidenori; Julien, Jean-Pierre; Obata, Nanako; Hosokawa, Masato; Akiyama, Haruhiko; Kaneko, Satoshi; Ayaki, Takashi; Ito, Hidefumi; Kaji, Ryuji; Takahashi, Ryosuke; Yamanaka, Shinya; Inoue, Haruhisa

2017-05-24

Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 ( SOD1 ). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein ( TDP-43 ) or repeat expansions in C9orf72 Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS. Copyright © 2017, American Association for the Advancement of Science.

DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection.

PubMed

Lindow, Janet C; Dohrmann, Paul R; McHenry, Charles S

2015-07-03

Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known β-binding domain, decreasing interaction with the β2 processivity factor. Surprisingly, mutations within the β binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Deletion of the γ-secretase subunits Aph1B/C impairs memory and worsens the deficits of knock-in mice modeling the Alzheimer-like familial Danish dementia

PubMed Central

Biundo, Fabrizio; Ishiwari, Keita; Del Prete, Dolores; D'Adamio, Luciano

2016-01-01

Mutations in BRI2/ITM2b genes cause Familial British and Danish Dementias (FBD and FDD), which are pathogenically similar to Familial Alzheimer Disease (FAD). BRI2 inhibits processing of Amyloid precursor protein (APP), a protein involved in FAD pathogenesis. Accumulation of a carboxyl-terminal APP metabolite –β-CTF- causes memory deficits in a knock-in mouse model of FDD, called FDDKI. We have investigated further the pathogenic function of β-CTF studying the effect of Aph1B/C deletion on FDDKI mice. This strategy is based on the evidence that deletion of Aph1B/C proteins, which are components of the γ-secretase that cleaves β-CTF, results in stabilization of β-CTF and a reduction of Aβ. We found that both the FDD mutation and the Aph1B/C deficiency mildly interfered with spatial long term memory, spatial working/short-term memory and long-term contextual fear memory. In addition, the Aph1BC deficiency induced deficits in long-term cued fear memory. Moreover, the two mutations have additive adverse effects as they compromise the accuracy of spatial long-term memory and induce spatial memory retention deficits in young mice. Overall, the data are consistent with a role for β-CTF in the genesis of memory deficits. PMID:26942869

Deletion of the γ-secretase subunits Aph1B/C impairs memory and worsens the deficits of knock-in mice modeling the Alzheimer-like familial Danish dementia.

PubMed

Biundo, Fabrizio; Ishiwari, Keita; Del Prete, Dolores; D'Adamio, Luciano

2016-03-15

Mutations in BRI2/ITM2b genes cause Familial British and Danish Dementias (FBD and FDD), which are pathogenically similar to Familial Alzheimer Disease (FAD). BRI2 inhibits processing of Amyloid precursor protein (APP), a protein involved in FAD pathogenesis. Accumulation of a carboxyl-terminal APP metabolite -ß-CTF- causes memory deficits in a knock-in mouse model of FDD, called FDDKI.We have investigated further the pathogenic function of ß-CTF studying the effect of Aph1B/C deletion on FDDKI mice. This strategy is based on the evidence that deletion of Aph1B/C proteins, which are components of the γ-secretase that cleaves ß-CTF, results in stabilization of ß-CTF and a reduction of Aβ. We found that both the FDD mutation and the Aph1B/C deficiency mildly interfered with spatial long term memory, spatial working/short-term memory and long-term contextual fear memory. In addition, the Aph1BC deficiency induced deficits in long-term cued fear memory. Moreover, the two mutations have additive adverse effects as they compromise the accuracy of spatial long-term memory and induce spatial memory retention deficits in young mice. Overall, the data are consistent with a role for β-CTF in the genesis of memory deficits.

Functional Analysis of AP-2 α and μ2 Subunits

PubMed Central

Motley, Alison M.; Berg, Nicola; Taylor, Marcus J.; Sahlender, Daniela A.; Hirst, Jennifer; Owen, David J.

2006-01-01

The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA–resistant α and μ2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of α, the phosphorylation site of μ2, or the YXXΦ binding site of μ2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of α, or mutating the PtdIns(4,5)P2 binding site of μ2, has no apparent effect. However, adding a C-terminal GFP tag to α renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo. PMID:17035630

APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane.

PubMed

Mills, Kate M; Brocardo, Mariana G; Henderson, Beric R

2016-02-01

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. © 2016 Mills et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, is a putative tumor suppressor.

PubMed

Kanno, Emiri; Kawasaki, Osamu; Takahashi, Kazuya; Takano, Kazunori; Endo, Takeshi

2018-01-01

Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf-MEK-ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae.

PubMed

Brenton, Ashley A; Souvannaseng, Lattha; Cheung, Kong; Anishchenko, Michael; Brault, Aaron C; Luckhart, Shirley

2014-06-23

Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance. We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae. The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK. MEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway.

Ehlers-Danlos Syndrome Type VI in a 17-Year-Old Iranian Boy with Severe Muscular Weakness – A Diagnostic Challenge?

PubMed Central

Kariminejad, Ariana; Bozorgmehr, Bita; Khatami, Alireza; Kariminejad, Mohamad-Hasan; Giunta, Cecilia; Steinmann, Beat

2010-01-01

Background The Ehlers-Danlos syndrome type VI (EDSVI) is an autosomal recessive connective tissue disease which is characterized by severe hypotonia at birth, progressive kyphoscoliosis, skin hyperelasticity and fragility, joint hypermobility and (sub-)luxations, microcornea, rupture of arteries and the eye globe, and osteopenia. The enzyme collagen lysyl hydroxylase (LH1) is deficient in these patients due to mutations in the PLOD1 gene. Case Presentation We report a 17-year-old boy, born to related parents, with severe kyphoscoliosis, scar formation, joint hypermobility and multiple dislocations, muscular weakness, rupture of an ocular globe, and a history of severe infantile hypotonia. EDS VI was suspected clinically and confirmed by an elevated ratio of urinary total lysyl pyridinoline to hydroxylysyl pyridinoline, abnormal electrophoretic mobility of the α-collagen chains, and mutation analysis. Conclusion Because of the high rate of consanguineous marriages in Iran and, as a consequence thereof, an increased rate of autosomal recessive disorders, we urge physicians to consider EDS VI in the differential diagnosis of severe infantile hypotonia and muscular weakness, a disorder which can easily be confirmed by the analysis of urinary pyridinolines that is highly specific, sensitive, robust, fast, non-invasive, and inexpensive. PMID:23056730

The USP8 mutational status may predict drug susceptibility in corticotroph adenomas of Cushing's disease.

PubMed

Hayashi, Kyohei; Inoshita, Naoko; Kawaguchi, Kohei; Ibrahim Ardisasmita, Arif; Suzuki, Hisanori; Fukuhara, Noriaki; Okada, Mitsuo; Nishioka, Hiroshi; Takeuchi, Yasuhiro; Komada, Masayuki; Takeshita, Akira; Yamada, Shozo

2016-02-01

Somatic mutations in the ubiquitin-specific peptidase USP8 gene were recently detected in one- to two-third(s) of corticotroph adenomas of Cushing's disease (CD). These mutations may lead to the deubiquitination of EGFR, thereby increasing EGFR signaling, which has been implicated in ACTH hypersecretion. Our objective was to determine the impact of USP8 mutations on the clinicopathological features of CD. USP8 mutations as well as clinicopathological characteristics were examined in 60 corticotroph adenomas including 15 Crooke's cell adenomas (CCAs), a rare histological variant presenting with generally aggressive behavior, using qRT-PCR and/or immunohistochemistry. USP8 mutations were exclusively detected in women, except for one case, with a prevalence of 42.2% in non-CCA and 13.3% in CCA (overall 35%). Clinically well-behaved presentations including microadenoma and curative resection were more common in mutated cases. The expression of EGFR was not associated with the mutation status. In contrast, mutated tumors expressed significantly higher levels of POMC, SSTR5, and MGMT. Microadenomas that strongly express POMC were common among mutated tumors, which may lead to the mechanisms by which very small adenomas secrete excess ACTH to present overt CD. While USP8 mutations were less likely to enhance tumorous ACTH hypersecretion via EGFR-mediated activation, the presence of USP8 mutations may predict favorable responses to the somatostatin analog pasireotide, which exhibits high affinity for SSTR5. In contrast, non-mutated aggressive tumors such as CCA may respond better to the alkylating agent temozolomide because of their significantly weak expression of MGMT. © 2016 European Society of Endocrinology.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

PubMed Central

2010-01-01

Background Immunohistochemistry (IHC) with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients. Methods EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC) cell lines and tumor samples from 78 stage IV NSCLC patients. Results IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA) deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93%) patients with exon 21 EGFR mutations (all with L858R) but did not identify the L861Q mutation in the remaining two patients. Conclusions IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients. PMID:21167064

Autosomal dominant distal myopathy due to a novel ACTA1 mutation.

PubMed

Liewluck, Teerin; Sorenson, Eric J; Walkiewicz, Magdalena A; Rumilla, Kandelaria M; Milone, Margherita

2017-08-01

Mutations in skeletal muscle α-actin 1-encoding gene (ACTA1) cause autosomal dominant or recessive myopathies with marked clinical and pathological heterogeneity. Patients typically develop generalized or limb-girdle pattern of weakness, but recently a family with scapuloperoneal myopathy was reported. We describe a father and 2 children with childhood-to-juvenile onset distal myopathy, carrying a novel dominant ACTA1 variant, c.757G>C (p.Gly253Arg). Father had delayed motor development and developed significant proximal weakness later in life; he was initially misdiagnosed as having spinal muscular atrophy based on electromyographic findings. His children had predominant anterior distal leg and finger extensor involvement. Nemaline rods were abundant on the daughter's biopsy, absent on the father's initial biopsy, and extremely rare on the father's subsequent biopsy a decade later. The father's second biopsy also showed myofibrillar pathology and rare fibers with actin filament aggregates. The present family expands the spectrum of actinopathy to include a distal myopathy. Copyright © 2017 Elsevier B.V. All rights reserved.

[Lower urinary tract dysfunction and neuropathological findings of the neural circuits controlling micturition in familial amyotrophic lateral sclerosis with L106V mutation in the SOD1 gene].

PubMed

Hineno, Akiyo; Oyanagi, Kiyomitsu; Nakamura, Akinori; Shimojima, Yoshio; Yoshida, Kunihiro; Ikeda, Shu-Ichi

2016-01-01

We report lower urinary tract dysfunction and neuropathological findings of the neural circuits controlling micturition in the patients with familial amyotrophic lateral sclerosis having L106V mutation in the SOD1 gene. Ten of 20 patients showed lower urinary tract dysfunction and 5 patients developed within 1 year after the onset of weakness. In 8 patients with an artificial respirator, 6 patients showed lower urinary tract dysfunction. Lower urinary tract dysfunction and respiratory failure requiring an artificial respirator occurred simultaneously in 3 patients. Neuronal loss and gliosis were observed in the neural circuits controlling micturition, such as frontal lobe, thalamus, hypothalamus, striatum, periaqueductal gray, ascending spinal tract, lateral corticospinal tract, intermediolateral nucleus and Onufrowicz' nucleus. Lower urinary tract dysfunction, especially storage symptoms, developed about 1 year after the onset of weakness, and the dysfunction occurred simultaneously with artificial respirator use in the patients.

Fixation Probability in a Haploid-Diploid Population

PubMed Central

Bessho, Kazuhiro; Otto, Sarah P.

2017-01-01

Classical population genetic theory generally assumes either a fully haploid or fully diploid life cycle. However, many organisms exhibit more complex life cycles, with both free-living haploid and diploid stages. Here we ask what the probability of fixation is for selected alleles in organisms with haploid-diploid life cycles. We develop a genetic model that considers the population dynamics using both the Moran model and Wright–Fisher model. Applying a branching process approximation, we obtain an accurate fixation probability assuming that the population is large and the net effect of the mutation is beneficial. We also find the diffusion approximation for the fixation probability, which is accurate even in small populations and for deleterious alleles, as long as selection is weak. These fixation probabilities from branching process and diffusion approximations are similar when selection is weak for beneficial mutations that are not fully recessive. In many cases, particularly when one phase predominates, the fixation probability differs substantially for haploid-diploid organisms compared to either fully haploid or diploid species. PMID:27866168

Memory Reactivation Predicts Resistance to Retroactive Interference: Evidence from Multivariate Classification and Pattern Similarity Analyses

PubMed Central

Rugg, Michael D.

2016-01-01

Memory reactivation—the reinstatement of processes and representations engaged when an event is initially experienced—is believed to play an important role in strengthening and updating episodic memory. The present study examines how memory reactivation during a potentially interfering event influences memory for a previously experienced event. Participants underwent fMRI during the encoding phase of an AB/AC interference task in which some words were presented twice in association with two different encoding tasks (AB and AC trials) and other words were presented once (DE trials). The later memory test required retrieval of the encoding tasks associated with each of the study words. Retroactive interference was evident for the AB encoding task and was particularly strong when the AC encoding task was remembered rather than forgotten. We used multivariate classification and pattern similarity analysis (PSA) to measure reactivation of the AB encoding task during AC trials. The results demonstrated that reactivation of generic task information measured with multivariate classification predicted subsequent memory for the AB encoding task regardless of whether interference was strong and weak (trials for which the AC encoding task was remembered or forgotten, respectively). In contrast, reactivation of neural patterns idiosyncratic to a given AB trial measured with PSA only predicted memory when the strength of interference was low. These results suggest that reactivation of features of an initial experience shared across numerous events in the same category, but not features idiosyncratic to a particular event, are important in resisting retroactive interference caused by new learning. SIGNIFICANCE STATEMENT Reactivating a previously encoded memory is believed to provide an opportunity to strengthen the memory, but also to return the memory to a labile state, making it susceptible to interference. However, there is debate as to how memory reactivation elicited by a potentially interfering event influences subsequent retrieval of the memory. The findings of the current study indicate that reactivating features idiosyncratic to a particular experience during interference only influences subsequent memory when interference is relatively weak. Critically, reactivation of generic contextual information predicts subsequent source memory when retroactive interference is either strong and weak. The results indicate that reactivation of generic information about a prior episode mitigates forgetting due to retroactive interference. PMID:27076433

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.

Quantifying stickiness: thermodynamic characterization of intramolecular domain interactions to guide the design of förster resonance energy transfer sensors.

PubMed

Lindenburg, Laurens H; Malisauskas, Mantas; Sips, Tari; van Oppen, Lisanne; Wijnands, Sjors P W; van de Graaf, Stan F J; Merkx, Maarten

2014-10-14

The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Förster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)-linker-enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (ΔG0(o-c) = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange.linker.mCherry fusion proteins yielded a toolbox of new mOrange-mCherry combinations that allowed tuning of their intramolecular interaction from very weak (ΔG0(o-c) = .0.39 kCal/mol) to relatively stable (ΔG0(o-c) = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean-Citrine pair by mOrange.mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategies.

Research and Implementation of Heart Sound Denoising

NASA Astrophysics Data System (ADS)

Liu, Feng; Wang, Yutai; Wang, Yanxiang

Heart sound is one of the most important signals. However, the process of getting heart sound signal can be interfered with many factors outside. Heart sound is weak electric signal and even weak external noise may lead to the misjudgment of pathological and physiological information in this signal, thus causing the misjudgment of disease diagnosis. As a result, it is a key to remove the noise which is mixed with heart sound. In this paper, a more systematic research and analysis which is involved in heart sound denoising based on matlab has been made. The study of heart sound denoising based on matlab firstly use the powerful image processing function of matlab to transform heart sound signals with noise into the wavelet domain through wavelet transform and decomposition these signals in muli-level. Then for the detail coefficient, soft thresholding is made using wavelet transform thresholding to eliminate noise, so that a signal denoising is significantly improved. The reconstructed signals are gained with stepwise coefficient reconstruction for the processed detail coefficient. Lastly, 50HZ power frequency and 35 Hz mechanical and electrical interference signals are eliminated using a notch filter.

Multiple Origins of a Mitochondrial Mutation Conferring Deafness

PubMed Central

Hutchin, T. P.; Cortopassi, G. A.

1997-01-01

A point mutation (1555G) in the smaller ribosomal subunit of the mitochondrial DNA (mtDNA) has been associated with maternally inherited traits of hypersensitivity to streptomycin and sensorineural deafness in a number of families from China, Japan, Israel, and Africa. To determine whether this distribution was the result of a single or multiple mutational events, we carried out genetic distance analysis and phylogenetic analysis of 10 independent mtDNA D-loop sequences from Africa and Asia. The mtDNA sequence diversity was high (2.21%). Phylogenetic analysis assigned 1555G-bearing haplotypes at very divergent points in the human mtDNA evolutionary tree, and the 1555G mutations occur in many cases on race-specific mtDNA haplotypes, both facts are inconsistent with a recent introgression of the mutation into these races. The simplest interpretation of the available data is that there have been multiple origins of the 1555G mutation. The genetic distance among mtDNAs bearing the pathogenic 1555G mutation is much larger than among mtDNAs bearing either evolutionarily neutral or weakly deleterious nucleotide substitutions (such as the 4336G mutation). These results are consistent with the view that pathogenic mtDNA haplotypes such as 1555G arise on disparate mtDNA lineages which because of negative natural selection leave relatively few related descendants. The co-existence of the same mutation with deafness in individuals with very different nuclear and mitochondrial genetic backgrounds confirms the pathogenicity of the 1555G mutation. PMID:9055086

Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

PubMed

Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

2016-09-01

Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Effective lethal mutagenesis of influenza virus by three nucleoside analogs.

PubMed

Pauly, Matthew D; Lauring, Adam S

2015-04-01

Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Novel germline PALB2 truncating mutations in African-American breast cancer patients

PubMed Central

Zheng, Yonglan; Zhang, Jing; Niu, Qun; Huo, Dezheng; Olopade, Olufunmilayo I.

2011-01-01

Background It has been demonstrated that PALB2 acts as a bridging molecule between the BRCA1 and BRCA2 proteins and is responsible for facilitating BRCA2-mediated DNA repair. Truncating mutations in the PALB2 gene have been reported to be enriched in Fanconi anemia and breast cancer patients in various populations. Methods We evaluated the contribution of PALB2 germline mutations in 279 African-American breast cancer patients including 29 patients with a strong family history, 29 patients with a moderate family history, 75 patients with a weak family history, and 146 non-familial or sporadic breast cancer cases. Results After direct sequencing of all the coding exons, exon/intron boundaries, 5′UTR and 3′UTR of PALB2, three (1.08%; 3 in 279) novel monoallelic truncating mutations were identified: c.758dupT (exon4), c.1479delC (exon4) and c.3048delT (exon 10); together with 50 sequence variants, 27 of which are novel. None of the truncating mutations were found in 262 controls from the same population. Conclusions PALB2 mutations are present in both familial and non-familial breast cancer among African-Americans. Rare PALB2 mutations account for a small but substantial proportion of breast cancer patients. PMID:21932393

Clinical Manifestation and a New "ISCU" Mutation in Iron-Sulphur Cluster Deficiency Myopathy

ERIC Educational Resources Information Center

Kollberg, Gittan; Tulinius, Mar; Melberg, Atle; Darin, Niklas; Andersen, Oluf; Holmgren, Daniel; Oldfors, Anders; Holme, Elisabeth

2009-01-01

Myopathy with deficiency of succinate dehydrogenase and aconitase is a recessively inherited disorder characterized by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, severe metabolic acidosis and rhabdomyolysis may occur. The…

CHARGE syndrome: a recurrent hotspot of mutations in CHD7 IVS25 analyzed by bioinformatic tools and minigene assays.

PubMed

Legendre, Marine; Rodriguez-Ballesteros, Montserrat; Rossi, Massimiliano; Abadie, Véronique; Amiel, Jeanne; Revencu, Nicole; Blanchet, Patricia; Brioude, Frédéric; Delrue, Marie-Ange; Doubaj, Yassamine; Sefiani, Abdelaziz; Francannet, Christine; Holder-Espinasse, Muriel; Jouk, Pierre-Simon; Julia, Sophie; Melki, Judith; Mur, Sébastien; Naudion, Sophie; Fabre-Teste, Jennifer; Busa, Tiffany; Stamm, Stephen; Lyonnet, Stanislas; Attie-Bitach, Tania; Kitzis, Alain; Gilbert-Dussardier, Brigitte; Bilan, Frédéric

2018-02-01

CHARGE syndrome is a rare genetic disorder mainly due to de novo and private truncating mutations of CHD7 gene. Here we report an intriguing hot spot of intronic mutations (c.5405-7G > A, c.5405-13G > A, c.5405-17G > A and c.5405-18C > A) located in CHD7 IVS25. Combining computational in silico analysis, experimental branch-point determination and in vitro minigene assays, our study explains this mutation hot spot by a particular genomic context, including the weakness of the IVS25 natural acceptor-site and an unconventional lariat sequence localized outside the common 40 bp upstream the acceptor splice site. For each of the mutations reported here, bioinformatic tools indicated a newly created 3' splice site, of which the existence was confirmed using pSpliceExpress, an easy-to-use and reliable splicing reporter tool. Our study emphasizes the idea that combining these two complementary approaches could increase the efficiency of routine molecular diagnosis.

A case of neuromyotonia and axonal motor neuropathy: A report of a HINT1 mutation in the United States.

PubMed

Jerath, Nivedita U; Shy, Michael E; Grider, Tiffany; Gutmann, Ludwig

2015-12-01

HINT1 mutations cause an autosomal recessive distal hereditary motor axonal neuropathy with neuromyotonia. This is a case report of a HINT1 mutation in the United States. A 30-year-old man of Slovenian heritage and no significant family history presented with scoliosis as a child and later developed neuromyotonia and distal weakness. Electrodiagnostic testing revealed an axonal motor neuropathy and neuromyotonic discharges. Previous diagnostic work-up, including testing for Cx32, MPZ, PMP-22, NF-L, EGR2, CLCN1, DM1, DM2, SMN exon 7/8, emerin, LMNA, MPK, SCNA4, acid maltase gene, paraneoplastic disorder, and a sural nerve biopsy, was negative. Genetic testing for a HINT1 mutation was performed and revealed a homozygous mutation at p.Arg37Pro. This entity should be distinguished clinically and genetically from myotonic dystrophy and channelopathies with the clinical features of neuromyotonia and an axonal neuropathy. This case illustrates the importance of identifying the correct phenotype to avoid unnecessary and costly evaluations. © 2015 Wiley Periodicals, Inc.

A chemical chaperone improves muscle function in mice with a RyR1 mutation.

PubMed

Lee, Chang Seok; Hanna, Amy D; Wang, Hui; Dagnino-Acosta, Adan; Joshi, Aditya D; Knoblauch, Mark; Xia, Yan; Georgiou, Dimitra K; Xu, Jianjun; Long, Cheng; Amano, Hisayuki; Reynolds, Corey; Dong, Keke; Martin, John C; Lagor, William R; Rodney, George G; Sahin, Ergun; Sewry, Caroline; Hamilton, Susan L

2017-03-24

Mutations in the RYR1 gene cause severe myopathies. Mice with an I4895T mutation in the type 1 ryanodine receptor/Ca 2+ release channel (RyR1) display muscle weakness and atrophy, but the underlying mechanisms are unclear. Here we show that the I4895T mutation in RyR1 decreases the amplitude of the sarcoplasmic reticulum (SR) Ca 2+ transient, resting cytosolic Ca 2+ levels, muscle triadin content and calsequestrin (CSQ) localization to the junctional SR, and increases endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and mitochondrial ROS production. Treatment of mice carrying the I4895T mutation with a chemical chaperone, sodium 4-phenylbutyrate (4PBA), reduces ER stress/UPR and improves muscle function, but does not restore SR Ca 2+ transients in I4895T fibres to wild type levels, suggesting that decreased SR Ca 2+ release is not the major driver of the myopathy. These findings suggest that 4PBA, an FDA-approved drug, has potential as a therapeutic intervention for RyR1 myopathies that are associated with ER stress.

A chemical chaperone improves muscle function in mice with a RyR1 mutation

PubMed Central

Lee, Chang Seok; Hanna, Amy D.; Wang, Hui; Dagnino-Acosta, Adan; Joshi, Aditya D.; Knoblauch, Mark; Xia, Yan; Georgiou, Dimitra K.; Xu, Jianjun; Long, Cheng; Amano, Hisayuki; Reynolds, Corey; Dong, Keke; Martin, John C.; Lagor, William R.; Rodney, George G.; Sahin, Ergun; Sewry, Caroline; Hamilton, Susan L.

2017-01-01

Mutations in the RYR1 gene cause severe myopathies. Mice with an I4895T mutation in the type 1 ryanodine receptor/Ca2+ release channel (RyR1) display muscle weakness and atrophy, but the underlying mechanisms are unclear. Here we show that the I4895T mutation in RyR1 decreases the amplitude of the sarcoplasmic reticulum (SR) Ca2+ transient, resting cytosolic Ca2+ levels, muscle triadin content and calsequestrin (CSQ) localization to the junctional SR, and increases endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and mitochondrial ROS production. Treatment of mice carrying the I4895T mutation with a chemical chaperone, sodium 4-phenylbutyrate (4PBA), reduces ER stress/UPR and improves muscle function, but does not restore SR Ca2+ transients in I4895T fibres to wild type levels, suggesting that decreased SR Ca2+ release is not the major driver of the myopathy. These findings suggest that 4PBA, an FDA-approved drug, has potential as a therapeutic intervention for RyR1 myopathies that are associated with ER stress. PMID:28337975

Potential Use of a Weak Ethylene Receptor Mutant, Sletr1-2, as Breeding Material To Extend Fruit Shelf Life of Tomato.

PubMed

Mubarok, Syariful; Okabe, Yoshihiro; Fukuda, Naoya; Ariizumi, Tohru; Ezura, Hiroshi

2015-09-16

Mutations in the ethylene receptor gene (SlETR1), Sletr1-1 and Sletr1-2, are effective in reducing ethylene sensitivity and improving fruit shelf life. In this study the effect of Sletr1-1 and Sletr1-2 mutations was investigated in F1 hybrid lines. These two mutants and control were crossed with four commercial pure-line tomatoes. The Sletr1-1 mutation showed undesirable pleiotropic effects in the F1 hybrid lines. The Sletr1-2 mutation was effective in improving fruit shelf life of F1 hybrid lines for 4-5 days longer. It was also effective in improving fruit firmness without change in fruit size, ethylene production, respiration rate, and total soluble solids or a great reduction in fruit color, lycopene, and β-carotene, although the titratable acidity was increased by Sletr1-2 mutation. These results indicate that the Sletr1-2 mutant allele has the potential to improve fruit shelf life via incorporation in tomato breeding programs.

Two Novel Mutations in the GDAP1 and PRX Genes in Early Onset Charcot-Marie-Tooth Syndrome

PubMed Central

Auer-Grumbach, M.; Fischer, C.; Papić, L.; John, E.; Plecko, B.; Bittner, R. E.; Bernert, G.; Pieber, T. R.; Miltenberger, G.; Schwarz, R.; Windpassinger, C.; Grill, F.; Timmerman, V.; Speicher, M. R.; Janecke, A. R.

2011-01-01

Autosomal recessive Charcot-Marie-Tooth syndrome (AR-CMT) is often characterised by an infantile disease onset and a severe phenotype. Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene are thought to be a common cause of AR-CMT. Mutations in the periaxin (PRX) gene are rare. They are associated with severe demyelination of the peripheral nerves and sometimes lead to prominent sensory disturbances. To evaluate the frequency of GDAP1 and PRX mutations in early onset CMT, we examined seven AR-CMT families and 12 sporadic CMT patients, all presenting with progressive distal muscle weakness and wasting. In one family also prominent sensory abnormalities and sensory ataxia were apparent from early childhood. In three families we detected four GDAP1 mutations (L58LfsX4, R191X, L239F and P153L), one of which is novel and is predicted to cause a loss of protein function. In one additional family with prominent sensory abnormalities a novel homozygous PRX mutation was found (A700PfsX17). No mutations were identified in 12 sporadic cases. This study suggests that mutations in the GDAP1 gene are a common cause of early-onset AR-CMT. In patients with early-onset demyelinating AR-CMT and severe sensory loss PRX is one of the genes to be tested. PMID:18504680

Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

NASA Technical Reports Server (NTRS)

Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

2002-01-01

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

Role of the Transcription Factor Sox4 in Insulin Secretion and Impaired Glucose Tolerance

PubMed Central

Goldsworthy, Michelle; Hugill, Alison; Freeman, Helen; Horner, Emma; Shimomura, Kenju; Bogani, Debora; Pieles, Guido; Mijat, Vesna; Arkell, Ruth; Bhattacharya, Shoumo; Ashcroft, Frances M.; Cox, Roger D.

2008-01-01

OBJECTIVES— To identify, map, clone, and functionally validate a novel mouse model for impaired glucose tolerance and insulin secretion. RESEARCH DESIGN AND METHODS— Haploinsufficiency of the insulin receptor and associated mild insulin resistance has been used to sensitize an N-ethyl-N-nitrosourea (ENU) screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. The new impaired glucose tolerance 4 (IGT4) model was selected using an intraperitoneal glucose tolerance test and inheritance of the phenotype confirmed by generation of backcross progeny. Segregation of the phenotype was correlated with genotype information to map the location of the gene and candidates sequenced for mutations. The function of the SRY-related high mobility group (HMG)-box 4 (Sox4) gene in insulin secretion was tested using another ENU allele and by small interfering RNA silencing in insulinoma cells. RESULTS— We describe two allelic autosomal dominant mutations in the highly conserved HMG box of the transcription factor Sox4. Previously associated with pancreas development, Sox4 mutations in the adult mouse result in an insulin secretory defect, which exhibits impaired glucose tolerance in association with insulin receptor+/−–induced insulin resistance. Elimination of the Sox4 transcript in INS1 and Min6 cells resulted in the abolition of glucose-stimulated insulin release similar to that observed for silencing of the key metabolic enzyme glucokinase. Intracellular calcium measurements in treated cells indicate that this defect lies downstream of the ATP-sensitive K+ channel (KATP channel) and calcium influx. CONCLUSIONS— IGT4 represents a novel digenic model of insulin resistance coupled with an insulin secretory defect. The Sox4 gene has a role in insulin secretion in the adult β-cell downstream of the KATP channel. PMID:18477811

Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function.

PubMed

Bjursell, Magnus K; Blom, Henk J; Cayuela, Jordi Asin; Engvall, Martin L; Lesko, Nicole; Balasubramaniam, Shanti; Brandberg, Göran; Halldin, Maria; Falkenberg, Maria; Jakobs, Cornelis; Smith, Desiree; Struys, Eduard; von Döbeln, Ulrika; Gustafsson, Claes M; Lundeberg, Joakim; Wedell, Anna

2011-10-07

Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

EEC- and ADULT-associated TP63 mutations exhibit functional heterogeneity toward P63 responsive sequences.

PubMed

Monti, Paola; Russo, Debora; Bocciardi, Renata; Foggetti, Giorgia; Menichini, Paola; Divizia, Maria T; Lerone, Margherita; Graziano, Claudio; Wischmeijer, Anita; Viadiu, Hector; Ravazzolo, Roberto; Inga, Alberto; Fronza, Gilberto

2013-06-01

TP63 germ-line mutations are responsible for a group of human ectodermal dysplasia syndromes, underlining the key role of P63 in the development of ectoderm-derived tissues. Here, we report the identification of two TP63 alleles, G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identified G134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized in yeast, studied in a mammalian cell line and modeled based on the crystal structure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showed both complete loss of transactivation function and ability to interfere over wild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63α-specific REs derived from genes closely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinct functional effect of each mutant. The present results highlight the importance of integrating different functional endpoints that take in account the features of P63 proteins' target sequences to examine the impact of TP63 mutations and the associated clinical variability. © 2013 Wiley Periodicals, Inc.

From animal models to human disease: a genetic approach for personalized medicine in ALS.

PubMed

Picher-Martel, Vincent; Valdmanis, Paul N; Gould, Peter V; Julien, Jean-Pierre; Dupré, Nicolas

2016-07-11

Amyotrophic Lateral Sclerosis (ALS) is the most frequent motor neuron disease in adults. Classical ALS is characterized by the death of upper and lower motor neurons leading to progressive paralysis. Approximately 10 % of ALS patients have familial form of the disease. Numerous different gene mutations have been found in familial cases of ALS, such as mutations in superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS), C9ORF72, ubiquilin-2 (UBQLN2), optineurin (OPTN) and others. Multiple animal models were generated to mimic the disease and to test future treatments. However, no animal model fully replicates the spectrum of phenotypes in the human disease and it is difficult to assess how a therapeutic effect in disease models can predict efficacy in humans. Importantly, the genetic and phenotypic heterogeneity of ALS leads to a variety of responses to similar treatment regimens. From this has emerged the concept of personalized medicine (PM), which is a medical scheme that combines study of genetic, environmental and clinical diagnostic testing, including biomarkers, to individualized patient care. In this perspective, we used subgroups of specific ALS-linked gene mutations to go through existing animal models and to provide a comprehensive profile of the differences and similarities between animal models of disease and human disease. Finally, we reviewed application of biomarkers and gene therapies relevant in personalized medicine approach. For instance, this includes viral delivering of antisense oligonucleotide and small interfering RNA in SOD1, TDP-43 and C9orf72 mice models. Promising gene therapies raised possibilities for treating differently the major mutations in familial ALS cases.

The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo.

PubMed

Monet, Marie; Domenga, Valérie; Lemaire, Barbara; Souilhol, Céline; Langa, Francina; Babinet, Charles; Gridley, Thomas; Tournier-Lasserve, Elisabeth; Cohen-Tannoudji, Michel; Joutel, Anne

2007-04-15

Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy.

Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin

PubMed Central

Hu, Li-Yen R.; Ackermann, Maegen A.; Hecker, Peter A.; Prosser, Benjamin L.; King, Brendan; O’Connell, Kelly A.; Grogan, Alyssa; Meyer, Logan C.; Berndsen, Christopher E.; Wright, Nathan T.; Jonathan Lederer, W.; Kontrogianni-Konstantopoulos, Aikaterini

2017-01-01

Obscurins are cytoskeletal proteins with structural and regulatory roles encoded by OBSCN. Mutations in OBSCN are associated with the development of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Specifically, the R4344Q mutation present in immunoglobulin domain 58 (Ig58) was the first to be linked with the development of HCM. To assess the effects of R4344Q in vivo, we generated the respective knock-in mouse model. Mutant obscurins are expressed and incorporated normally into sarcomeres. The expression patterns of sarcomeric and Ca2+-cycling proteins are unaltered in sedentary 1-year-old knock-in myocardia, with the exception of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase 2 (SERCA2) and pentameric phospholamban whose levels are significantly increased and decreased, respectively. Isolated cardiomyocytes from 1-year-old knock-in hearts exhibit increased Ca2+-transients and Ca2+-load in the sarcoplasmic reticulum and faster contractility kinetics. Moreover, sedentary 1-year-old knock-in animals develop tachycardia accompanied by premature ventricular contractions, whereas 2-month-old knock-in animals subjected to pressure overload develop a DCM-like phenotype. Structural analysis revealed that the R4344Q mutation alters the distribution of electrostatic charges over the Ig58 surface, thus interfering with its binding capabilities. Consistent with this, wild-type Ig58 interacts with phospholamban modestly, and this interaction is markedly enhanced in the presence of R4344Q. Together, our studies demonstrate that under sedentary conditions, the R4344Q mutation results in Ca2+ deregulation and spontaneous arrhythmia, whereas in the presence of chronic, pathological stress, it leads to cardiac remodeling and dilation. We postulate that enhanced binding between mutant obscurins and phospholamban leads to SERCA2 disinhibition, which may underlie the observed pathological alterations. PMID:28630914

Semi-dwarfism and lodging tolerance in tef (Eragrostis tef) is linked to a mutation in the α-Tubulin 1 gene

PubMed Central

Jöst, Moritz; Esfeld, Korinna; Burian, Agata; Cannarozzi, Gina; Chanyalew, Solomon; Kuhlemeier, Cris; Assefa, Kebebew; Tadele, Zerihun

2015-01-01

Genetic improvement of native crops is a new and promising strategy to combat hunger in the developing world. Tef is the major staple food crop for approximately 50 million people in Ethiopia. As an indigenous cereal, it is well adapted to diverse climatic and soil conditions; however, its productivity is extremely low mainly due to susceptibility to lodging. Tef has a tall and weak stem, liable to lodge (or fall over), which is aggravated by wind, rain, or application of nitrogen fertilizer. To circumvent this problem, the first semi-dwarf lodging-tolerant tef line, called kegne, was developed from an ethyl methanesulphonate (EMS)-mutagenized population. The response of kegne to microtubule-depolymerizing and -stabilizing drugs, as well as subsequent gene sequencing and segregation analysis, suggests that a defect in the α-Tubulin gene is functionally and genetically tightly linked to the kegne phenotype. In diploid species such as rice, homozygous mutations in α-Tubulin genes result in extreme dwarfism and weak stems. In the allotetraploid tef, only one homeologue is mutated, and the presence of the second intact α-Tubulin gene copy confers the agriculturally beneficial semi-dwarf and lodging-tolerant phenotype. Introgression of kegne into locally adapted and popular tef cultivars in Ethiopia will increase the lodging tolerance in the tef germplasm and, as a result, will improve the productivity of this valuable crop. PMID:25399019

Genotoxicity testing of different types of beverages in the Drosophila wing Somatic Mutation And Recombination Test.

PubMed

Graf, U; Moraga, A A; Castro, R; Díaz Carrillo, E

1994-05-01

Five wines and one brandy of Spanish origin as well as three herbal teas and ordinary black tea were tested for genotoxicity in the wing Somatic Mutation And Recombination Test (SMART) which makes use of the two recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) on the left arm of chromosome 3 of Drosophila melanogaster. 3-day-old larvae trans-heterozygous for these two markers were fed the beverages at different concentrations and for different feeding periods using Drosophila instant medium. Somatic mutations or mitotic recombinations induced in the cells of the wing imaginal discs give rise to mutant single or twin spots on the wing blade of the emerging adult flies showing either the mwh phenotype or/and the flr phenotype. One of the red wines showed a clear genotoxic activity that was not due to its ethanol content. Two herbal teas (Urtica dioica, Achillea millefolium) and black tea (Camellia sinensis) proved to be weakly genotoxic as well. Furthermore, it was shown that quercetin and rutin, two flavonols present in beverages of plant origin, also exhibited weak genotoxic activity in the somatic cells of Drosophila. These results demonstrate that Drosophila in vivo somatic assays can detect the genotoxicity of complex mixtures such as beverages. In particular, it is possible to administer these test materials in the same form as that in which they are normally consumed.

Fluctuating Selection in the Moran

PubMed Central

Dean, Antony M.; Lehman, Clarence; Yi, Xiao

2017-01-01

Contrary to classical population genetics theory, experiments demonstrate that fluctuating selection can protect a haploid polymorphism in the absence of frequency dependent effects on fitness. Using forward simulations with the Moran model, we confirm our analytical results showing that a fluctuating selection regime, with a mean selection coefficient of zero, promotes polymorphism. We find that increases in heterozygosity over neutral expectations are especially pronounced when fluctuations are rapid, mutation is weak, the population size is large, and the variance in selection is big. Lowering the frequency of fluctuations makes selection more directional, and so heterozygosity declines. We also show that fluctuating selection raises dn/ds ratios for polymorphism, not only by sweeping selected alleles into the population, but also by purging the neutral variants of selected alleles as they undergo repeated bottlenecks. Our analysis shows that randomly fluctuating selection increases the rate of evolution by increasing the probability of fixation. The impact is especially noticeable when the selection is strong and mutation is weak. Simulations show the increase in the rate of evolution declines as the rate of new mutations entering the population increases, an effect attributable to clonal interference. Intriguingly, fluctuating selection increases the dn/ds ratios for divergence more than for polymorphism, a pattern commonly seen in comparative genomics. Our model, which extends the classical neutral model of molecular evolution by incorporating random fluctuations in selection, accommodates a wide variety of observations, both neutral and selected, with economy. PMID:28108586

Mitochondrial-nuclear interactions and accelerated compensatory evolution: evidence from the primate cytochrome C oxidase complex.

PubMed

Osada, Naoki; Akashi, Hiroshi

2012-01-01

Accelerated rates of mitochondrial protein evolution have been proposed to reflect Darwinian coadaptation for efficient energy production for mammalian flight and brain activity. However, several features of mammalian mtDNA (absence of recombination, small effective population size, and high mutation rate) promote genome degradation through the accumulation of weakly deleterious mutations. Here, we present evidence for "compensatory" adaptive substitutions in nuclear DNA- (nDNA) encoded mitochondrial proteins to prevent fitness decline in primate mitochondrial protein complexes. We show that high mutation rate and small effective population size, key features of primate mitochondrial genomes, can accelerate compensatory adaptive evolution in nDNA-encoded genes. We combine phylogenetic information and the 3D structure of the cytochrome c oxidase (COX) complex to test for accelerated compensatory changes among interacting sites. Physical interactions among mtDNA- and nDNA-encoded components are critical in COX evolution; amino acids in close physical proximity in the 3D structure show a strong tendency for correlated evolution among lineages. Only nuclear-encoded components of COX show evidence for positive selection and adaptive nDNA-encoded changes tend to follow mtDNA-encoded amino acid changes at nearby sites in the 3D structure. This bias in the temporal order of substitutions supports compensatory weak selection as a major factor in accelerated primate COX evolution.

A novel canine model for Duchenne muscular dystrophy (DMD): single nucleotide deletion in DMD gene exon 20.

PubMed

Mata López, Sara; Hammond, James J; Rigsby, Madison B; Balog-Alvarez, Cynthia J; Kornegay, Joe N; Nghiem, Peter P

2018-05-29

Boys with Duchenne muscular dystrophy (DMD) have DMD gene mutations, with associated loss of the dystrophin protein and progressive muscle degeneration and weakness. Corticosteroids and palliative support are currently the best treatment options. The long-term benefits of recently approved compounds such as eteplirsen and ataluren remain to be seen. Dogs with naturally occurring dystrophinopathies show progressive disease akin to that of DMD. Accordingly, canine DMD models are useful for studies of pathogenesis and preclinical therapy development. A dystrophin-deficient, male border collie dog was evaluated at the age of 5 months for progressive muscle weakness and dysphagia. Dramatically increased serum creatine kinase levels (41,520 U/L; normal range 59-895 U/L) were seen on a biochemistry panel. Histopathologic changes characteristic of dystrophinopathy were seen. Dystrophin was absent in the skeletal muscle on immunofluorescence microscopy and western blot. Whole genome sequencing, polymerase chain reaction, and Sanger sequencing revealed a frameshift, single nucleotide deletion in canine DMD exon 20, position 27,626,466 (c.2841delT mRNA), resulting in a stop codon six nucleotides downstream. Semen was archived for future line perpetuation. This spontaneous canine dystrophinopathy occurred due to a novel mutation in the minor DMD mutation hotspot (between exons 2 through 20). Perpetuating this line could allow for preclinical testing of genetic therapies targeted to this area of the DMD gene.

A source of artifact in the lacZ reversion assay in Escherichia coli.

PubMed

Hoffmann, George R; Gray, Carol L; Lange, Paulina B; Marando, Christie I

2015-06-01

The lacZ reversion assay in Escherichia coli measures point mutations that occur by specific base substitutions and frameshift mutations. The tester strains cannot use lactose as a carbon source (Lac(-)), and revertants are easily detected by growth on lactose medium (Lac(+)). Six strains identify the six possible base substitutions, and five strains measure +G, -G, -CG, +A and -A frameshifts. Strong mutagens give dose-dependent increases in numbers of revertants per plate and revertant frequencies. Testing compounds that are arguably nonmutagens or weakly mutagenic, we often noted statistically significant dose-dependent increases in revertant frequency that were not accompanied by an absolute increase in numbers of revertants. The increase in frequency was wholly ascribable to a declining number of viable cells owing to toxicity. Analysis of the conditions revealed that the frequency of spontaneous revertants is higher when there are fewer viable cells per plate. The phenomenon resembles "adaptive" or "stress" mutagenesis, whereby lactose revertants accumulate in Lac(-) bacteria under starvation conditions in the absence of catabolite repression. Adaptive mutation is observed after long incubation and might be expected to be irrelevant in a standard assay using 48-h incubation. However, we found that elevated revertant frequencies occur under typical assay conditions when the bacterial lawn is thin, and this can cause increases in revertant frequency that mimic chemical mutagenesis when treatments are toxic but not mutagenic. Responses that resemble chemical mutagenesis were observed in the absence of mutagenic treatment in strains that revert by different frameshift mutations. The magnitude of the artifact is affected by cell density, dilution, culture age, incubation time, catabolite repression and the age and composition of media. Although the specific reversion assay is effective for quickly distinguishing classes of mutations induced by potent mutagens, its utility for discerning effects of weak mutagens may be compromised by the artifact. Copyright © 2015 Elsevier B.V. All rights reserved.

Newly identified CHO ERCC3/XPB mutations and phenotype characterization

PubMed Central

Rybanská, Ivana; Gurský, Ján; Fašková, Miriam; Salazar, Edmund P.; Kimlíčková-Polakovičová, Erika; Kleibl, Karol; Thompson, Larry H.; Piršel, Miroslav

2010-01-01

Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER. PMID:19942596

Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

PubMed Central

Cline, Susan D.

2012-01-01

How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

Epidemiology of echinocandin resistance in Candida

PubMed Central

Grossman, Nina T.; Chiller, Tom M.; Lockhart, Shawn R.

2018-01-01

Echinocandins are the newest antifungal agents approved for use in treating Candida infections in the US. They act by interfering with 1,3-β-D-glucan synthase and therefore disrupt cell wall production and lead to Candida cell death. There is no intrinsic resistance to echinocandins among Candida species, and isolates from historic collections archived before the release of the echinocandins show no resistance. Resistance to the echinocandins remains low among most Candida species and ranges overall from 0–1%. Among isolates of Candida glabrata, the proportion of resistant isolates is higher and has been reported to be as high as 13.5% in at least one hospital. Antifungal resistance is due to specific amino acid mutations in the Fksp subunit(s) of the 1,3-β-D-glucan synthase protein which are localized to one of two hotspots. These mutations are being recognized in isolates from patients who have failed echinocandin therapy, and often lead to a poor outcome. While the future looks bright for the echinocandins against most Candida species, C. glabrata remains a species of concern and resistance rates of C. glabrata to the echinocandins should be monitored closely. PMID:29780439

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

PubMed

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-05-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib

PubMed Central

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-01-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC. PMID:28521430

Deletion of a Single-Copy Trna Affects Microtubule Function in Saccharomyces Cerevisiae

PubMed Central

Reijo, R. A.; Cho, D. S.; Huffaker, T. C.

1993-01-01

rts1-1 was identified as an extragenic suppressor of tub2-104, a cold-sensitive allele of the sole gene encoding β-tubulin in the yeast, Saccharomyces cerevisiae. In addition, rts1-1 cells are heat sensitive and resistant to the microtubule-destabilizing drug, benomyl. The rts1-1 mutation is a deletion of approximately 5 kb of genomic DNA on chromosome X that includes one open reading frame and three tRNA genes. Dissection of this region shows that heat sensitivity is due to deletion of the open reading frame (HIT1). Suppression and benomyl resistance are caused by deletion of the gene encoding a tRNA(AGG)(Arg) (HSX1). Northern analysis of rts1-1 cells indicates that HSX1 is the only gene encoding this tRNA. Deletion of HSX1 does not suppress the tub2-104 mutation by misreading at the AGG codons in TUB2. It also does not suppress by interfering with the protein arginylation that targets certain proteins for degradation. These results leave open the prospect that this tRNA(AGG)(Arg) plays a novel role in the cell. PMID:8307335

Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness.

PubMed

Wang, Yang; Melkani, Girish C; Suggs, Jennifer A; Melkani, Anju; Kronert, William A; Cammarato, Anthony; Bernstein, Sanford I

2012-06-01

Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

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

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

First description of phosphofructokinase deficiency in spain: identification of a novel homozygous missense mutation in the PFKM gene

PubMed Central

Vives-Corrons, Joan-Lluis; Koralkova, Pavla; Grau, Josep M.; Mañú Pereira, Maria del Mar; Van Wijk, Richard

2013-01-01

Phosphofructokinase deficiency is a very rare autosomal recessive disorder, which belongs to group of rare inborn errors of metabolism called glycogen storage disease. Here we report on a new mutation in the phosphofructokinase (PFK) gene PFKM identified in a 65-years-old woman who suffered from lifelong intermittent muscle weakness and painful spasms of random occurrence, episodic dark urines, and slight haemolytic anemia. After ruling out the most common causes of chronic haemolytic anemia, the study of a panel of 24 enzyme activities showed a markedly decreased PFK activity in red blood cells (RBCs) from the patient. DNA sequence analysis of the PFKM gene subsequently revealed a novel homozygous mutation: c.926A>G; p.Asp309Gly. This mutation is predicted to severely affect enzyme catalysis thereby accounting for the observed enzyme deficiency. This case represents a prime example of classical PFK deficiency and is the first reported case of this very rare red blood cell disorder in Spain. PMID:24427140

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

DOE PAGES

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.; ...

2018-02-14

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

[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.

Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis.

PubMed

Perez, Rodney H; Sugino, Haruki; Ishibashi, Naoki; Zendo, Takeshi; Wilaipun, Pongtep; Leelawatcharamas, Vichien; Nakayama, Jiro; Sonomoto, Kenji

2017-04-01

Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

EGFR-L858R mutant enhances lung adenocarcinoma cell invasive ability and promotes malignant pleural effusion formation through activation of the CXCL12-CXCR4 pathway

PubMed Central

Tsai, Meng-Feng; Chang, Tzu-Hua; Wu, Shang-Gin; Yang, Hsiao-Yin; Hsu, Yi-Chiung; Yang, Pan-Chyr; Shih, Jin-Yuan

2015-01-01

Malignant pleural effusion (MPE) is a common clinical problem in non-small cell lung carcinoma (NSCLC) patients; however, the underlying mechanisms are still largely unknown. Recent studies indicate that the frequency of the L858R mutant form of the epidermal growth factor receptor (EGFR-L858R) is higher in lung adenocarcinoma with MPE than in surgically resected specimens, suggesting that lung adenocarcinoma cells harboring this mutation tend to invade the adjacent pleural cavity. The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to investigate the molecular mechanisms involved in the formation of MPE. We found that expression of EGFR-L858R in lung cancer cells resulted in up-regulation of the CXCR4 in association with increased cancer cell invasive ability and MPE formation. Ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We also indicated that Inhibition of CXCR4 with small interfering RNA, neutralizing antibody, or receptor antagonist significantly suppressed the EGFR-L858R–dependent cell invasion. These results suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring a specific type of EGFR mutation. PMID:26338423

A KCNH2 branch point mutation causing aberrant splicing contributes to an explanation of genotype-negative long QT syndrome.

PubMed

Crotti, Lia; Lewandowska, Marzena A; Schwartz, Peter J; Insolia, Roberto; Pedrazzini, Matteo; Bussani, Erica; Dagradi, Federica; George, Alfred L; Pagani, Franco

2009-02-01

Genetic screening of long QT syndrome (LQTS) fails to identify disease-causing mutations in about 30% of patients. So far, molecular screening has focused mainly on coding sequence mutations or on substitutions at canonical splice sites. The purpose of this study was to explore the possibility that intronic variants not at canonical splice sites might affect splicing regulatory elements, lead to aberrant transcripts, and cause LQTS. Molecular screening was performed through DHPLC and sequence analysis. The role of the intronic mutation identified was assessed with a hybrid minigene splicing assay. A three-generation LQTS family was investigated. Molecular screening failed to identify an obvious disease-causing mutation in the coding sequences of the major LQTS genes but revealed an intronic A-to-G substitution in KCNH2 (IVS9-28A/G) cosegregating with the clinical phenotype in family members. In vitro analysis proved that the mutation disrupts the acceptor splice site definition by affecting the branch point (BP) sequence and promoting intron retention. We further demonstrated a tight functional relationship between the BP and the polypyrimidine tract, whose weakness is responsible for the pathological effect of the IVS9-28A/G mutation. We identified a novel BP mutation in KCNH2 that disrupts the intron 9 acceptor splice site definition and causes LQT2. The present finding demonstrates that intronic mutations affecting pre-mRNA processing may contribute to the failure of traditional molecular screening in identifying disease-causing mutations in LQTS subjects and offers a rationale strategy for the reduction of genotype-negative cases.

Argonaute2 is the catalytic engine of mammalian RNAi.

PubMed

Liu, Jidong; Carmell, Michelle A; Rivas, Fabiola V; Marsden, Carolyn G; Thomson, J Michael; Song, Ji-Joon; Hammond, Scott M; Joshua-Tor, Leemor; Hannon, Gregory J

2004-09-03

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

Glucose metabolism in gastric cancer: The cutting-edge

PubMed Central

Yuan, Lian-Wen; Yamashita, Hiroharu; Seto, Yasuyuki

2016-01-01

Glucose metabolism in gastric cancer cells differs from that of normal epithelial cells. Upregulated aerobic glycolysis (Warburg effect) in gastric cancer meeting the demands of cell proliferation is associated with genetic mutations, epigenetic modification and proteomic alteration. Understanding the mechanisms of aerobic glycolysis may contribute to our knowledge of gastric carcinogenesis. Metabolomic studies offer novel, convenient and practical tools in the search for new biomarkers for early detection, diagnosis, prognosis, and chemosensitivity prediction of gastric cancer. Interfering with the process of glycolysis in cancer cells may provide a new and promising therapeutic strategy for gastric cancer. In this article, we present a brief review of recent studies of glucose metabolism in gastric cancer, with primary focus on the clinical applications of new biomarkers and their potential therapeutic role in gastric cancer. PMID:26877609

A Population Genetic Model for the Initial Spread of Partially Resistant Malaria Parasites under Anti-Malarial Combination Therapy and Weak Intrahost Competition

PubMed Central

Kim, Yuseob; Escalante, Ananias A.; Schneider, Kristan A.

2014-01-01

To develop public-health policies that extend the lifespan of affordable anti-malarial drugs as effective treatment options, it is necessary to understand the evolutionary processes leading to the origin and spread of mutations conferring drug resistance in malarial parasites. We built a population-genetic model for the emergence of resistance under combination drug therapy. Reproductive cycles of parasites are specified by their absolute fitness determined by clinical parameters, thus coupling the evolutionary-genetic with population-dynamic processes. Initial mutations confer only partial drug-resistance. Therefore, mutant parasites rarely survive combination therapy and within-host competition is very weak among parasites. The model focuses on the early phase of such unsuccessful recurrent mutations. This ends in the rare event of mutants enriching in an infected individual from which the successful spread of resistance over the entire population is initiated. By computer simulations, the waiting time until the establishment of resistant parasites is analysed. Resistance spreads quickly following the first appearance of a host infected predominantly by mutant parasites. This occurs either through a rare transmission of a resistant parasite to an uninfected host or through a rare failure of drugs in removing “transient” mutant alleles. The emergence of resistance is delayed with lower mutation rate, earlier treatment, higher metabolic cost of resistance, longer duration of high drug dose, and higher drug efficacy causing a stronger reduction in the sensitive and resistant parasites’ fitnesses. Overall, contrary to other studies’ proposition, the current model based on absolute fitness suggests that aggressive drug treatment delays the emergence of drug resistance. PMID:25007207

Myotonia permanens with Nav1.4-G1306E displays varied phenotypes during course of life.

PubMed

Lehmann-Horn, Frank; D'Amico, Adele; Bertini, Enrico; Lomonaco, Mauro; Merlini, Luciano; Nelson, Kevin R; Philippi, Heike; Siciliano, Gabriele; Spaans, Frank; Jurkat-Rott, Karin

2017-09-01

Myotonia permanens due to Nav1.4-G1306E is a rare sodium channelopathy with potentially life-threatening respiratory complications. Our goal was to study phenotypic variability throughout life. Clinical neurophysiology and genetic analysis were performed. Using existing functional expression data we determined the sodium window by integration. In 10 unrelated patients who were believed to have epilepsy, respiratory disease or Schwartz-Jampel syndrome, we made the same prima facie diagnosis and detected the same heterologous Nav1.4-G1306E channel mutation as for our first myotonia permanens patient published in 1993. Eight mutations were de-novo, two were inherited from the affected parent each. Seven patients improved with age, one had a benign phenotype from birth, and two died of respiratory complications. The clinical features age-dependently varied with severe neonatal episodic laryngospasm in childhood and myotonia throughout life. Weakness of varying degrees was present. The responses to cold, exercise and warm-up were different for lower than for upper extremities. Spontaneous membrane depolarization increased frequency and decreased size of action potentials; self-generated repolarization did the opposite. The overlapping of steady-state activation and inactivation curves generated a 3.1-fold window area for G1306E vs. normal channels. Residue G1306 Neonatal laryngospasm and unusual distribution of myotonia, muscle hypertrophy, and weakness encourage direct search for the G1306E mutation, a hotspot for de-novo mutations. Successful therapy with the sodium channel blocker flecainide is due to stabilization of the inactivated state and special effectiveness for enlarged window currents. Our G1306E collection is the first genetically clarified case series from newborn period to adulthood and therefore helpful for counselling.

Myotonia permanens with Nav1.4-G1306E displays varied phenotypes during course of life

PubMed Central

LEHMANN-HORN, FRANK; D’AMICO, ADELE; BERTINI, ENRICO; LOMONACO, MAURO; MERLINI, LUCIANO; NELSON, KEVIN R.; PHILIPPI, HEIKE; SICILIANO, GABRIELE; SPAANS, FRANK; JURKAT-ROTT, KARIN

2017-01-01

Introduction Myotonia permanens due to Nav1.4-G1306E is a rare sodium channelopathy with potentially life-threatening respiratory complications. Our goal was to study phenotypic variability throughout life. Methods Clinical neurophysiology and genetic analysis were performed. Using existing functional expression data we determined the sodium window by integration. Results In 10 unrelated patients who were believed to have epilepsy, respiratory disease or Schwartz-Jampel syndrome, we made the same prima facie diagnosis and detected the same heterologous Nav1.4-G1306E channel mutation as for our first myotonia permanens patient published in 1993. Eight mutations were de-novo, two were inherited from the affected parent each. Seven patients improved with age, one had a benign phenotype from birth, and two died of respiratory complications. The clinical features age-dependently varied with severe neonatal episodic laryngospasm in childhood and myotonia throughout life. Weakness of varying degrees was present. The responses to cold, exercise and warm-up were different for lower than for upper extremities. Spontaneous membrane depolarization increased frequency and decreased size of action potentials; self-generated repolarization did the opposite. The overlapping of steady-state activation and inactivation curves generated a 3.1-fold window area for G1306E vs. normal channels. Discussion Residue G1306 Neonatal laryngospasm and unusual distribution of myotonia, muscle hypertrophy, and weakness encourage direct search for the G1306E mutation, a hotspot for de-novo mutations. Successful therapy with the sodium channel blocker flecainide is due to stabilization of the inactivated state and special effectiveness for enlarged window currents. Our G1306E collection is the first genetically clarified case series from newborn period to adulthood and therefore helpful for counselling. PMID:29774303

Genetic and Early Clinical Manifestations of Females Heterozygous for Duchenne/Becker Muscular Dystrophy.

PubMed

Papa, Riccardo; Madia, Francesca; Bartolomeo, Domenico; Trucco, Federica; Pedemonte, Marina; Traverso, Monica; Broda, Paolo; Bruno, Claudio; Zara, Federico; Minetti, Carlo; Fiorillo, Chiara

2016-02-01

Female carriers of Duchenne muscular dystrophy (DMD), although usually asymptomatic, develop muscle weakness up to 17% of the time, and a third present cardiac abnormalities or cognitive impairment. Clinical features of DMD carriers during childhood are poorly known. We describe a cohort of pediatric DMD carriers, providing clinical, genetic, and histopathologic features, with a mean follow-up of 7 years. Fifteen females with a DMD mutation (age range 5 to 18 years) were included. Seven patients (46%) presented with clinically evident symptoms and signs such as limb girdle weakness, abnormal gait, and exercise intolerance. The other eight patients (53%) were evaluated because of an incidental finding of elevated level of creatine kinase. Creatine kinase level was elevated in all, ranging from 392 to 13,000 U/L. Calf hypertrophy was observed in eight patients (53%). No patient developed respiratory or cardiac involvement. The most frequent complication was scoliosis (46%). Four patients (29%) also presented minor learning disabilities or behavioral problems. We performed electromyography in half of patients, showing myopathic pattern in four (53%). Muscle biopsy revealed a mosaic reduction of dystrophin in nine available cases. DMD gene mutations were mostly deletions (71%), resulting in loss of reading frame in five patients (36%). The three patients who experienced the most severe disease course were affected either by a nonsense or frameshift mutation. Our analysis suggests that DMD gene mutations may be suspected in a female child with persistently elevated levels of creatine kinase. Evidence of scoliosis, calf hypertrophy, or myopathic pattern at electromyography may also be helpful, and muscle biopsy is always indicative. DMD carriers should be followed for subtle orthopedic and psychiatric complications during childhood. Copyright © 2016 Elsevier Inc. All rights reserved.

Language Development in Individuals with Fragile X Syndrome

ERIC Educational Resources Information Center

Finestack, Lizbeth H.; Richmond, Erica K.; Abbeduto, Leonard

2009-01-01

Fragile X syndrome (FXS) is the leading inherited cause of intellectual disability. The syndrome is caused by a single gene mutation on the X chromosome. Although individual differences are large, most individuals with FXS display weaknesses across all language and literacy domains compared with peers of the same chronological age with typical…

Fixation Probability in a Haploid-Diploid Population.

PubMed

Bessho, Kazuhiro; Otto, Sarah P

2017-01-01

Classical population genetic theory generally assumes either a fully haploid or fully diploid life cycle. However, many organisms exhibit more complex life cycles, with both free-living haploid and diploid stages. Here we ask what the probability of fixation is for selected alleles in organisms with haploid-diploid life cycles. We develop a genetic model that considers the population dynamics using both the Moran model and Wright-Fisher model. Applying a branching process approximation, we obtain an accurate fixation probability assuming that the population is large and the net effect of the mutation is beneficial. We also find the diffusion approximation for the fixation probability, which is accurate even in small populations and for deleterious alleles, as long as selection is weak. These fixation probabilities from branching process and diffusion approximations are similar when selection is weak for beneficial mutations that are not fully recessive. In many cases, particularly when one phase predominates, the fixation probability differs substantially for haploid-diploid organisms compared to either fully haploid or diploid species. Copyright © 2017 by the Genetics Society of America.

Variable phenotypic expression and onset in MYH14 distal hereditary motor neuropathy phenotype in a large, multigenerational North American family.

PubMed

Iyadurai, Stanley; Arnold, W David; Kissel, John T; Ruhno, Corey; Mcgovern, Vicki L; Snyder, Pamela J; Prior, Thomas W; Roggenbuck, Jennifer; Burghes, Arthur H; Kolb, Stephen J

2017-08-01

Distal hereditary motor neuropathy (dHMN) causes distal-predominant weakness without prominent sensory loss. Myosin heavy chain disorders most commonly result in distal myopathy and cardiomyopathy with or without hearing loss, but a complex phenotype with dHMN, myopathy, hoarseness, and hearing loss was reported in a Korean family with a c.2822G>T mutation in MYH14. In this study we report phenotypic features in a North American family with the c.2822G>T in MYH14. Clinical and molecular characterization was performed in a large, 6-generation, Caucasian family with MYH14 dHMN. A total of 11 affected and 7 unaffected individuals were evaluated and showed varying age of onset and severity of weakness. Genotypic concordance was confirmed with molecular analysis. Electrophysiological studies demonstrated distal motor axonal degeneration without myopathy in all affected subjects tested. Mutation of MYH14 can result in a range of neuromuscular phenotypes that includes a dHMN and hearing loss phenotype with variable age of onset. Muscle Nerve 56: 341-345, 2017. © 2016 Wiley Periodicals, Inc.

Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes

PubMed Central

Dhar, Alok; Polev, Dmitrii E.; Masharsky, Alexey E.; Rogozin, Igor B.; Pavlov, Youri I.

2015-01-01

Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells. PMID:25941824

The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

PubMed Central

Zuurbier, Linda; Petricoin, Emanuel F.; Vuerhard, Maartje J.; Calvert, Valerie; Kooi, Clarissa; Buijs-Gladdines, Jessica G.C.A.M.; Smits, Willem K.; Sonneveld, Edwin; Veerman, Anjo J.P.; Kamps, Willem A.; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

2012-01-01

Background PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKT mutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1-activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors. PMID:22491738

An evolutionary reduction principle for mutation rates at multiple Loci.

PubMed

Altenberg, Lee

2011-06-01

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

Multi-institutional oncogenic driver mutation analysis in lung adenocarcinoma: The Lung Cancer Mutation Consortium experience

PubMed Central

Dias-Santagata, Dora; Wistuba, Ignacio I.; Chen, Heidi; Fujimoto, Junya; Kugler, Kelly; Franklin, Wilbur A.; Iafrate, A. John; Ladanyi, Marc; Kris, Mark G.; Johnson, Bruce E.; Bunn, Paul A.; Minna, John D.; Kwiatkowski, David J.

2015-01-01

Introduction Molecular genetic analyses of lung adenocarcinoma have recently become standard of care for treatment selection. The Lung Cancer Mutation Consortium was formed to enable collaborative multi-institutional analyses of 10 potential oncogenic driver mutations. Technical aspects of testing, and clinicopathologic correlations are presented. Methods Mutation testing in at least one of 8 genes (EGFR, KRAS, ERBB2, AKT1, BRAF, MEK1, NRAS, PIK3CA) using SNaPshot, mass spectrometry, Sanger sequencing +/− PNA and/or sizing assays, along with ALK and/or MET FISH were performed in 6 labs on 1007 patients from 14 institutions. Results 1007 specimens had mutation analysis performed, and 733 specimens had all 10 genes analyzed. Mutation identification rates did not vary by analytic method. Biopsy and cytology specimens were inadequate for testing in 26% and 35% of cases compared to 5% of surgical specimens. Among the 1007 cases with mutation analysis performed, EGFR, KRAS, ALK, and ERBB2 alterations were detected in 22, 25, 8.5, and 2.4% of cases, respectively. EGFR mutations were highly associated with female sex, Asian race, and never smoking status; and less strongly associated with stage IV disease, presence of bone metastases, and absence of adrenal metastases. ALK rearrangements were strongly associated with never smoking status, and more weakly associated with presence of liver metastases. ERBB2 mutations were strongly associated with Asian race and never smoking status. Two mutations were seen in 2.7% of samples, all but one of which involved one or more of PIK3CA, ALK or MET. Conclusion Multi-institutional molecular analysis across multiple platforms, sample types, and institutions can yield consistent results and novel clinicopathological observations. PMID:25738220

Flotation separation and electrothermal atomic absorption spectrometric determination of thallium in wastewater samples.

PubMed

Hosseini, Mohammad Saeid; Chamsaz, Mahmoud; Raissi, Heidar; Naseri, Yousef

2006-01-01

The proposed method is a simple process for the determination of trace amount of thallium(I) in the environmental wastewater samples by electrothermal atomic absorption spectrometry. The wastewater samples were obtained from the environment of a cement plant and subjected to a simple treatment, such as adjusting pH and masking the interfering ions, to prepare for the flotation process in which the thallium(I) content was floated as an ion-association complex using iodide and Rhodamine B at the interface of aqueous/cyclohexane layers. The floated layer was then separated and dissolved in 2 ml of a solution, which was 1% to H2SO4 and 50% to methanol, respectively. Aliquots of 10-microl of this solution were subjected to the graphite furnace to determine the thallium(I) content. The flotation process can be carried in a weak acidic medium in which the interfering effects owing to certain metal ions were eliminated by masking them as neutral citrate chelates. The dynamic range for the determination was found to be 1.0 x 10(-8) - 1.0 x 10(-7) mol l(-1). The RSD was 3.2% and the DL was 2.5 x 10(-9) mol l(-1) (calculated as 3SD of the blank). The reliability of the method is demonstrated by the analysis of a synthetic wastewater in which the recovery was found to be 94%.

Do parabens have the ability to interfere with steroidogenesis?

PubMed

Taxvig, Camilla; Vinggaard, Anne Marie; Hass, Ulla; Axelstad, Marta; Boberg, Julie; Hansen, Pernille Reimer; Frederiksen, Hanne; Nellemann, Christine

2008-11-01

The effects of ethyl and butyl paraben on steroidogenesis were evaluated in rats exposed in utero. Pregnant Wistar rats were dosed from gestational day (GD) 7 to GD 21, followed by examination of the dams, and the fetuses. Additionally, both parabens were tested in vitro in the H295R steroidogenesis assay and in the T-screen assay, the later to test for their ability to act as thyroid hormone receptor agonist or antagonist. In the in utero exposure toxicity study, neither ethyl nor butyl paraben showed any treatment-related effects on testosterone production, anogenital distance, or testicular histopathology. However, butyl paraben caused a significant decrease in the mRNA expression level of estradiol receptor-beta in fetal ovaries, and also significantly decreased the mRNA expression of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the adrenal glands. In vitro butyl paraben increased the proliferation of the GH3 cells in the T-Screen assay, thereby acting as a weak thyroid hormone receptor agonist. In the adrenal H295R steroidogenesis assay both ethyl and butyl paraben caused a significant increase in the progesterone formation. Overall, the results indicate that butyl paraben might have the ability to act as endocrine disruptor by interfering with the transport of cholesterol to the mitochondrion, thereby interfering with steroidogenesis, but also that the two tested parabens do not show clear endocrine disrupting capabilities in our short-term in vivo experiment.

Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy.

PubMed

Miyake, Noriko; Fukai, Ryoko; Ohba, Chihiro; Chihara, Takahiro; Miura, Masayuki; Shimizu, Hiroshi; Kakita, Akiyoshi; Imagawa, Eri; Shiina, Masaaki; Ogata, Kazuhiro; Okuno-Yuguchi, Jiu; Fueki, Noboru; Ogiso, Yoshifumi; Suzumura, Hiroshi; Watabe, Yoshiyuki; Imataka, George; Leong, Huey Yin; Fattal-Valevski, Aviva; Kramer, Uri; Miyatake, Satoko; Kato, Mitsuhiro; Okamoto, Nobuhiko; Sato, Yoshinori; Mitsuhashi, Satomi; Nishino, Ichizo; Kaneko, Naofumi; Nishiyama, Akira; Tamura, Tomohiko; Mizuguchi, Takeshi; Nakashima, Mitsuko; Tanaka, Fumiaki; Saitsu, Hirotomo; Matsumoto, Naomichi

2016-10-06

We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and β-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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

Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko

Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important rolemore » of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.« less

Functional correction of dystrophin actin binding domain mutations by genome editing

PubMed Central

Kyrychenko, Viktoriia; Kyrychenko, Sergii; Tiburcy, Malte; Shelton, John M.; Long, Chengzu; Schneider, Jay W.; Zimmermann, Wolfram-Hubertus; Bassel-Duby, Rhonda

2017-01-01

Dystrophin maintains the integrity of striated muscles by linking the actin cytoskeleton with the cell membrane. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that result in progressive, debilitating muscle weakness, cardiomyopathy, and a shortened lifespan. Mutations of dystrophin that disrupt the amino-terminal actin-binding domain 1 (ABD-1), encoded by exons 2–8, represent the second-most common cause of DMD. In the present study, we compared three different strategies for CRISPR/Cas9 genome editing to correct mutations in the ABD-1 region of the DMD gene by deleting exons 3–9, 6–9, or 7–11 in human induced pluripotent stem cells (iPSCs) and by assessing the function of iPSC-derived cardiomyocytes. All three exon deletion strategies enabled the expression of truncated dystrophin protein and restoration of cardiomyocyte contractility and calcium transients to varying degrees. We show that deletion of exons 3–9 by genomic editing provides an especially effective means of correcting disease-causing ABD-1 mutations. These findings represent an important step toward eventual correction of common DMD mutations and provide a means of rapidly assessing the expression and function of internally truncated forms of dystrophin-lacking portions of ABD-1. PMID:28931764

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

PubMed Central

Houlleberghs, Hellen; Dekker, Marleen; Lantermans, Hildo; Kleinendorst, Roos; Dubbink, Hendrikus Jan; Hofstra, Robert M. W.; Verhoef, Senno; te Riele, Hein

2016-01-01

Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that “oligo targeting” can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors. PMID:26951660

Mutation-selection equilibrium in games with mixed strategies.

PubMed

Tarnita, Corina E; Antal, Tibor; Nowak, Martin A

2009-11-07

We develop a new method for studying stochastic evolutionary game dynamics of mixed strategies. We consider the general situation: there are n pure strategies whose interactions are described by an nxn payoff matrix. Players can use mixed strategies, which are given by the vector (p(1),...,p(n)). Each entry specifies the probability to use the corresponding pure strategy. The sum over all entries is one. Therefore, a mixed strategy is a point in the simplex S(n). We study evolutionary dynamics in a well-mixed population of finite size. Individuals reproduce proportional to payoff. We consider the case of weak selection, which means the payoff from the game is only a small contribution to overall fitness. Reproduction can be subject to mutation; a mutant adopts a randomly chosen mixed strategy. We calculate the average abundance of every mixed strategy in the stationary distribution of the mutation-selection process. We find the crucial conditions that specify if a strategy is favored or opposed by selection. One condition holds for low mutation rate, another for high mutation rate. The result for any mutation rate is a linear combination of those two. As a specific example we study the Hawk-Dove game. We prove general statements about the relationship between games with pure and with mixed strategies.

A new titinopathy

PubMed Central

De Cid, Rafael; Ben Yaou, Rabah; Roudaut, Carinne; Charton, Karine; Baulande, Sylvain; Leturcq, France; Romero, Norma Beatriz; Malfatti, Edoardo; Beuvin, Maud; Vihola, Anna; Criqui, Audrey; Nelson, Isabelle; Nectoux, Juliette; Ben Aim, Laurène; Caloustian, Christophe; Olaso, Robert; Udd, Bjarne; Bonne, Gisèle; Eymard, Bruno

2015-01-01

Objective: To identify the genetic defects present in 3 families with muscular dystrophy, contractures, and calpain 3 deficiency. Methods: We performed targeted exome sequencing on one patient presenting a deficiency in calpain 3 on Western blot but for which mutations in the gene had been excluded. The identification of a homozygous truncating mutation in the M-line part of titin prompted us to sequence this region in 2 additional patients presenting similar clinical and biochemical characteristics. Results: The 3 patients shared similar features: coexistence of limb-girdle weakness and early-onset diffuse joint contractures without cardiomyopathy. The biopsies showed rimmed vacuoles, a dystrophic pattern, and secondary reduction in calpain 3. We identified a novel homozygous mutation in the exon Mex3 of the TTN gene in the first patient. At protein level, this mutation introduces a stop codon at the level of Mex3. Interestingly, we identified truncating mutations in both alleles in the same region of the TTN gene in patients from 2 additional families. Molecular protein analyses confirm loss of the C-ter part of titin. Conclusions: Our study broadens the phenotype of titinopathies with the report of a new clinical entity with prominent contractures and no cardiac abnormality and where the recessive mutations lead to truncation of the M-line titin and secondary calpain 3 deficiency. PMID:26581302

Overlapping SETBP1 gain-of-function mutations in Schinzel-Giedion syndrome and hematologic malignancies

PubMed Central

Steehouwer, Marloes; Gilissen, Christian; Graham, Sarah A.; Hoover-Fong, Julie; Telegrafi, Aida B.; Destree, Anne; Smigiel, Robert; Lambie, Lindsday A.; Kayserili, Hülya; Altunoglu, Umut; Lapi, Elisabetta; Uzielli, Maria Luisa; Aracena, Mariana; Nur, Banu G.; Mihci, Ercan; Moreira, Lilia M. A.; Borges Ferreira, Viviane; Horovitz, Dafne D. G.; da Rocha, Katia M.; Jezela-Stanek, Aleksandra; Brooks, Alice S.; Reutter, Heiko; Cohen, Julie S.; Fatemi, Ali; Smitka, Martin; Grebe, Theresa A.; Di Donato, Nataliya; Deshpande, Charu; Vandersteen, Anthony; Marques Lourenço, Charles; Dufke, Andreas; Rossier, Eva; Andre, Gwenaelle; Baumer, Alessandra; Spencer, Careni; McGaughran, Julie; Franke, Lude; Veltman, Joris A.; De Vries, Bert B. A.; Schinzel, Albert; Fisher, Simon E.; Hoischen, Alexander

2017-01-01

Schinzel-Giedion syndrome (SGS) is a rare developmental disorder characterized by multiple malformations, severe neurological alterations and increased risk of malignancy. SGS is caused by de novo germline mutations clustering to a 12bp hotspot in exon 4 of SETBP1. Mutations in this hotspot disrupt a degron, a signal for the regulation of protein degradation, and lead to the accumulation of SETBP1 protein. Overlapping SETBP1 hotspot mutations have been observed recurrently as somatic events in leukemia. We collected clinical information of 47 SGS patients (including 26 novel cases) with germline SETBP1 mutations and of four individuals with a milder phenotype caused by de novo germline mutations adjacent to the SETBP1 hotspot. Different mutations within and around the SETBP1 hotspot have varying effects on SETBP1 stability and protein levels in vitro and in in silico modeling. Substitutions in SETBP1 residue I871 result in a weak increase in protein levels and mutations affecting this residue are significantly more frequent in SGS than in leukemia. On the other hand, substitutions in residue D868 lead to the largest increase in protein levels. Individuals with germline mutations affecting D868 have enhanced cell proliferation in vitro and higher incidence of cancer compared to patients with other germline SETBP1 mutations. Our findings substantiate that, despite their overlap, somatic SETBP1 mutations driving malignancy are more disruptive to the degron than germline SETBP1 mutations causing SGS. Additionally, this suggests that the functional threshold for the development of cancer driven by the disruption of the SETBP1 degron is higher than for the alteration in prenatal development in SGS. Drawing on previous studies of somatic SETBP1 mutations in leukemia, our results reveal a genotype-phenotype correlation in germline SETBP1 mutations spanning a molecular, cellular and clinical phenotype. PMID:28346496

Limb Girdle Muscular Dystrophy Type 2E Due to a Novel Large Deletion in SGCB Gene.

PubMed

Ghafouri-Fard, Soudeh; Hashemi-Gorji, Feyzollah; Fardaei, Majid; Miryounesi, Mohammad

2017-01-01

Autosomal recessive limb-girdle muscular dystrophies (LGMD type 2) are a group of clinically and genetically heterogeneous diseases with the main characteristics of weakness and wasting of the pelvic and shoulder girdle muscles. Among them are sarcoglycanopathies caused by mutations in at least four genes named SGCA, SGCB, SGCG and SGCD. Here we report a consanguineous Iranian family with two children affected with LGMD type 2E. Mutation analysis revealed a novel homozygous exon 2 deletion of SGCB gene in the patients with the parents being heterozygous for this deletion. This result presents a novel underlying genetic mechanism for LGMD type 2E.

A novel de novo germ-line V292M mutation in the extracellular region of RET in a patient with phaeochromocytoma and medullary thyroid carcinoma: functional characterization.

PubMed

Castellone, Maria D; Verrienti, Antonella; Magendra Rao, Deva; Sponziello, Marialuisa; Fabbro, Dora; Muthu, Magesh; Durante, Cosimo; Maranghi, Marianna; Damante, Giuseppe; Pizzolitto, Stefano; Costante, Giuseppe; Russo, Diego; Santoro, Massimo; Filetti, Sebastiano

2010-10-01

In multiple endocrine neoplasia (MEN), rearranged during transfection (RET), gene testing has been extensively exploited to characterize tumour aggressiveness and optimize the diagnostic and clinical management. To report the underlying genetic alterations in an unusual case of MEN type 2 (MEN-2A). Occult medullary thyroid carcinoma (MTC) was diagnosed in a 44-year-old man who had presented with unilateral phaeochromcytoma. DNA extracted from the blood and tumour tissues was analysed for mutations in RET. The transforming potential and mitogenic properties of the identified RET mutation were investigated. The patient carried a novel heterozygous germ-line RET mutation in exon 5 (Val292Met, GTG>ATG) (V292M/RET) with no evidence of additional somatic alterations. The mutation maps to the third cadherin-like domain of RET, which is usually not included in RET screening. Interestingly, MTC with concomitant phaeochromcytoma has never been associated with a RET mutation involving the extracellular cadherin-like domain. V292M/RET was absent in the only two relatives examined. In vitro assays indicate that the mutant has low-grade transforming potential. Complete characterization and classification of all novel RET mutations are essential for extending genetic analysis in clinical practice. Our findings suggest that: (i) in all MEN-2 patients negative for RET hot-spot mutations, testing should be extended to all coding regions of the gene and (ii) the newly identified V292M/RET mutation is characterized by relatively weak in vitro transforming ability. © 2010 Blackwell Publishing Ltd.

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

PubMed Central

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

2012-01-01

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

Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila.

PubMed

White-Cooper, H; Carmena, M; Gonzalez, C; Glover, D M

1996-11-01

We have simultaneously screened for new alleles and second site mutations that fail to complement five cell cycle mutations of Drosphila carried on a single third chromosome (gnu, polo, mgr, asp, stg). Females that are either transheterozygous for scott of the antartic (scant) and polo, or homozygous for scant produce embryos that show mitotic defects. A maternal effect upon embryonic mitoses is also seen in embryos derived from females transheterozygous with helter skelter (hsk) and either mgr or asp. cleopatra (cleo), fails to complement asp but is not uncovered by a deficiency for asp. The mitotic phenotype of larvae heterozygous for cleo and the multiple mutant chromosome is similar to weak alleles of asp, but there are no defects in male meiosis. Mutations that failed to complement stg fell into two complementation groups corresponding to stg and a new gene noose. Three of the new stg alleles are early zygotic lethals, whereas the fourth is a pharate adult lethal allele that affects both mitosis and meiosis. Mutations in noose fully complement a small deficiency that removes stg, but when placed in trans to certain stg alleles, result in late lethality and mitotic abnormalities in larval brains.

Familial solitary chondrosarcoma resulting from germline EXT2 mutation.

PubMed

Heddar, Abdelkader; Fermey, Pierre; Coutant, Sophie; Angot, Emilie; Sabourin, Jean-Christophe; Michelin, Paul; Parodi, Nathalie; Charbonnier, Françoise; Vezain, Myriam; Bougeard, Gaëlle; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tournier, Isabelle

2017-02-01

Germline mutations of EXT2, encoding Exostosin Glycosyltransferase 2, are associated with multiple osteochondromas (MO), an autosomal dominant disease characterized by the development of multiple peripheral cartilaginous benign tumors with a weak risk of malignant transformation. We report here a family with a remarkable clinical presentation characterized by the development of isolated chondrosarcomas, mostly located in ribs. Comparative analysis of exomes from two third-degree affected relatives led us to identify a single common disruptive variation, corresponding to a stop mutation (c.237G > A, p.Trp79*; (NM_000401.3); c.138G > A, p.Trp46*; (NM_207122.1)) within exon 2 of the EXT2 gene. Interestingly, no obvious sign of MO was detected in affected members by radiological examination. This report shows that germline mutations of EXT2 can result, not only in the development of multiple benign osteochondromas, but also in the development of isolated malignant cartilaginous tumors including central tumors, and that the presence of germline EXT2 mutation should be considered in patients suspected to have an inherited predisposition to chondrosarcoma, even in the absence of MO. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Expression Profile Analysis of Zinc Transporters (ZIP4, ZIP9, ZIP11, ZnT9) in Gliomas and their Correlation with IDH1 Mutation Status.

PubMed

Kang, Xing; Chen, Rong; Zhang, Jie; Li, Gang; Dai, Peng-Gao; Chen, Chao; Wang, Hui-Juan

2015-01-01

Zinc transporters have been considered as essential regulators in many cancers; however, their mechanisms remain unknown, especially in gliomas. Isocitrate dehydrogenase 1(IDH1) mutation is crucial to glioma. This study aimed to investigate whether zinc transporters are correlated with glioma grade and IDH1 mutation status. IDH1 mutation status and mRNA expression of four zinc transporters (ZIP4, ZIP9, ZIP11, and ZnT9) were determined by subjecting a panel of 74 glioma tissue samples to quantitative real-time PCR and pyrosequencing. The correlations between the expression levels of these zinc transporter genes and the grade of glioma, as well as IDH1 mutation status, were investigated. Among the four zinc transporter genes, high ZIP4 expression and low ZIP11 expression were significantly associated with higher grade (grades III and IV) tumors compared with lower grade (grades I and II) counterparts (p<0.0001). However, only ZIP11 exhibited weak correlation with IDH1 mutation status (p=0.045). Samples with mutations in IDH1 displayed higher ZIP11 expression than those without IDH1 mutations. This finding indicated that zinc transporters may interact with IDH1 mutation by direct modulation or action in some shared pathways or genes to promote the development of glioma. Zinc transporters may play an important role in glioma. ZIP4 and ZIP11 are promising molecular diagnostic markers and novel therapeutic targets. Nevertheless, the detailed biological function of zinc transporters and the mechanism of the potential interaction between ZIP11 and IDH1 mutation in gliomagenesis should be further investigated.

Fluctuating Selection in the Moran.

PubMed

Dean, Antony M; Lehman, Clarence; Yi, Xiao

2017-03-01

Contrary to classical population genetics theory, experiments demonstrate that fluctuating selection can protect a haploid polymorphism in the absence of frequency dependent effects on fitness. Using forward simulations with the Moran model, we confirm our analytical results showing that a fluctuating selection regime, with a mean selection coefficient of zero, promotes polymorphism. We find that increases in heterozygosity over neutral expectations are especially pronounced when fluctuations are rapid, mutation is weak, the population size is large, and the variance in selection is big. Lowering the frequency of fluctuations makes selection more directional, and so heterozygosity declines. We also show that fluctuating selection raises d n / d s ratios for polymorphism, not only by sweeping selected alleles into the population, but also by purging the neutral variants of selected alleles as they undergo repeated bottlenecks. Our analysis shows that randomly fluctuating selection increases the rate of evolution by increasing the probability of fixation. The impact is especially noticeable when the selection is strong and mutation is weak. Simulations show the increase in the rate of evolution declines as the rate of new mutations entering the population increases, an effect attributable to clonal interference. Intriguingly, fluctuating selection increases the d n / d s ratios for divergence more than for polymorphism, a pattern commonly seen in comparative genomics. Our model, which extends the classical neutral model of molecular evolution by incorporating random fluctuations in selection, accommodates a wide variety of observations, both neutral and selected, with economy. Copyright © 2017 by the Genetics Society of America.

PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

PubMed

Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2015-10-15

Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

Probing electron delays in above-threshold ionization

DOE PAGES

Zipp, Lucas J.; Natan, Adi; Bucksbaum, Philip H.

2014-11-21

Recent experiments have revealed attosecond delays in the emission of electrons from atoms ionized by extreme UV light, offering a glimpse into the ultrafast nature of light-induced electron dynamics. In this work, we extend these measurements to the strong-field above-threshold ionization (ATI) regime, by measuring delays in the photoemission of electrons from argon in the presence of an intense laser field. We probe the ATI process with a weak coherent reference, at half the laser frequency. The interfering ionization signal reveals the relative spectral phase of adjacent ATI channels, with an equivalent resolution of a few attoseconds. These relative delaysmore » depend on the strong field, and approach zero at higher intensity. Our phase measurements of ATI electrons show how strong fields alter ionization dynamics in atoms.« less

Investigation of Poor Academic Achievement in Children with Duchenne Muscular Dystrophy

ERIC Educational Resources Information Center

Hinton, V. J.; De Vivo, D. C.; Fee, R.; Goldstein, E.; Stern, Y.

2004-01-01

Duchenne Muscular Dystrophy (DMD) is a neurogenetic developmental disorder that presents with progressive muscular weakness. It is caused by a mutation in a gene that results in the absence of specific products that normally localize to muscle cells and the central nervous system (CNS). The majority of affected individuals have IQs within the…

Clinical features and ryanodine receptor type 1 gene mutation analysis in a Chinese family with central core disease.

PubMed

Chang, Xingzhi; Jin, Yiwen; Zhao, Haijuan; Huang, Qionghui; Wang, Jingmin; Yuan, Yun; Han, Ying; Qin, Jiong

2013-03-01

Central core disease is a rare inherited neuromuscular disorder caused by mutations in ryanodine receptor type 1 gene. The clinical phenotype of the disease is highly variable. We report a Chinese pedigree with central core disease confirmed by the gene sequencing. All 3 patients in the family presented with mild proximal limb weakness. The serum level of creatine kinase was normal, and electromyography suggested myogenic changes. The histologic analysis of muscle biopsy showed identical central core lesions in almost all of the muscle fibers in the index case. Exon 90-106 in the C-terminal domain of the ryanodine receptor type 1 gene was amplified using polymerase chain reaction. One heterozygous missense mutation G14678A (Arg4893Gln) in exon 102 was identified in all 3 patients. This is the first report of a familial case of central core disease confirmed by molecular study in mainland China.

Physical Model for the Evolution of the Genetic Code

NASA Astrophysics Data System (ADS)

Yamashita, Tatsuro; Narikiyo, Osamu

2011-12-01

Using the shape space of codons and tRNAs we give a physical description of the genetic code evolution on the basis of the codon capture and ambiguous intermediate scenarios in a consistent manner. In the lowest dimensional version of our description, a physical quantity, codon level is introduced. In terms of the codon levels two scenarios are typically classified into two different routes of the evolutional process. In the case of the ambiguous intermediate scenario we perform an evolutional simulation implemented cost selection of amino acids and confirm a rapid transition of the code change. Such rapidness reduces uncomfortableness of the non-unique translation of the code at intermediate state that is the weakness of the scenario. In the case of the codon capture scenario the survival against mutations under the mutational pressure minimizing GC content in genomes is simulated and it is demonstrated that cells which experience only neutral mutations survive.

A novel PNPLA2 mutation causes neutral lipid storage disease with myopathy (NLSDM) presenting muscular dystrophic features with lipid storage and rimmed vacuoles.

PubMed

Chen, J; Hong, D; Wang, Z; Yuan, Y

2010-01-01

Neutral lipid storage disease with myopathy (NLSDM) is a type of lipid storage myopathy arising due to a mutation in the PNPLA2 gene encoding an adipose triglyceride lipase responsible for the degradation of intracellular triglycerides. Herein, we report the cases of two siblings manifesting slowly progressive proximal and distal limb weakness in adulthood. One of the patients had dilated cardiomyopathy, hearing loss and short stature. Muscle specimens of the 2 patients revealed muscular dystrophic features with massive lipid droplets and numerous rimmed vacuoles in the fibers. A novel homozygous mutation IVS2+1G > A in the PNPLA2 gene was identified in the 2 cases, but not in the healthy familial individuals. The presence of massive lipid droplets with muscular dystrophic changes and rimmed vacuoles in muscle fibers might be one of the characteristic pathological changes of NLSDM.

A case report: Becker muscular dystrophy presenting with epilepsy and dysgnosia induced by duplication mutation of Dystrophin gene.

PubMed

Miao, Jing; Feng, Jia-Chun; Zhu, Dan; Yu, Xue-Fan

2016-12-12

Becker muscular dystrophy (BMD), a genetic disorder of X-linked recessive inheritance, typically presents with gradually progressive muscle weakness. The condition is caused by mutations of Dystrophin gene located at Xp21.2. Epilepsy is an infrequent manifestation of BMD, while cases of BMD with dysgnosia are extremely rare. We describe a 9-year-old boy with BMD, who presented with epilepsy and dysgnosia. Serum creatine kinase level was markedly elevated (3665 U/L). Wechsler intelligence tests showed a low intelligence quotient (IQ = 65). Electromyogram showed slight myogenic changes and skeletal muscle biopsy revealed muscular dystrophy. Immunohistochemical staining showed partial positivity of sarcolemma for dystrophin-N. Multiplex ligation-dependent probe amplification revealed a duplication mutation in exons 37-44 in the Dystrophin gene. The present case report helps to better understand the clinical and genetic features of BMD.

Emotional responses to work-family conflict: an examination of gender role orientation working men and women.

PubMed

Livingston, Beth A; Judge, Timothy A

2008-01-01

The present study tested the effect of work-family conflict on emotions and the moderating effects of gender role orientation. On the basis of a multilevel design, the authors found that family-interfering-with- work was positively related to guilt, and gender role orientation interacted with both types of conflict (work-interfering-with-family and family-interfering-with-work) to predict guilt. Specifically, in general, traditional individuals experienced more guilt from family-interfering-with-work, and egalitarian individuals experienced more guilt from work-interfering-with-family. Additionally, a higher level interaction indicated that traditional men tended to experience a stronger relationship between family-interfering-with-work and guilt than did egalitarian men or women of either gender role orientation. 2008 APA

An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

PubMed

Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

2017-10-01

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis.

PubMed

Niemietz, Christoph; Chandhok, Gursimran; Schmidt, Hartmut

2015-09-30

The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR), expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP). Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO) and small interfering RNA (siRNA) designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

Natural disease history of mouse models for limb girdle muscular dystrophy types 2D and 2F

PubMed Central

Putker, K.; Tanganyika-de Winter, C. L.; Boertje-van der Meulen, J. W.; van Vliet, L.; Overzier, M.; Plomp, J. J.; Aartsma-Rus, A.; van Putten, M.

2017-01-01

Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F mice. PMID:28797108

Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.

PubMed

Rentsendorj, Otgonchimeg; Nagy, Andrea; Sinkó, Ildikó; Daraba, Andreea; Barta, Endre; Kiss, Ibolya

2005-08-01

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.

Early-onset severe hereditary sensory and autonomic neuropathy type 1 with S331F SPTLC1 mutation.

PubMed

Suh, Bum Chun; Hong, Young Bin; Nakhro, Khriezhanuo; Nam, Soo Hyun; Chung, Ki Wha; Choi, Byung-Ok

2014-02-01

Hereditary sensory and autonomic neuropathy type I (HSAN I) is an autosomal dominant disease characterized by prominent sensory impairment, resulting in foot ulcers or amputations and has a juvenile to adult onset. The major underlying causes of HSAN I are mutations in SPTLC1, which encodes the first subunit of serine palmitoyltransferase (SPT). To date, there have been no reports with regard to an HSAN patient of Korean origin. In this report we discussed an HSAN I patient with a missense mutation in SPTLC1 (c.992C>T: p.S331F). The patient had noticed frequent falls, lower leg weakness and hand tremors at age five. The patient also presented with foot ulcers, muscle hypotrophy, cataracts, hoarseness, vocal cord palsy and respiratory difficulties and succumbed to the condition at the age of 28 years. In accordance with previous reports, a mutation in Ser331 in the present patient was associated with early-onset and a severe phenotype. Therefore, Ser331 in SPTLC1 is a crucial amino acid, which characterizes the HSAN I phenotype.

Mitochondrial myopathies.

PubMed

DiMauro, Salvatore

2006-11-01

Our understanding of mitochondrial diseases (defined restrictively as defects of the mitochondrial respiratory chain) is expanding rapidly. In this review, I will give the latest information on disorders affecting predominantly or exclusively skeletal muscle. The most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency and mutations in genes controlling mitochondrial DNA abundance and structure, such as POLG, TK2, and MPV17. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with decreased amount and altered structure of cardiolipin, the main phospholipid of the inner mitochondrial membrane, but a secondary impairment of respiratory chain function is plausible. The role of mutations in protein-coding genes of mitochondrial DNA in causing isolated myopathies has been confirmed. Mutations in tRNA genes of mitochondrial DNA can also cause predominantly myopathic syndromes and--contrary to conventional wisdom--these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, cramps, recurrent myoglobinuria, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

Distinct distal myopathy phenotype caused by VCP gene mutation in a Finnish family.

PubMed

Palmio, Johanna; Sandell, Satu; Suominen, Tiina; Penttilä, Sini; Raheem, Olayinka; Hackman, Peter; Huovinen, Sanna; Haapasalo, Hannu; Udd, Bjarne

2011-08-01

Inclusion body myopathy with Paget disease and frontotemporal dementia (IBMPFD) is caused by mutations in the valosin-containing protein (VCP) gene. We report a new distal phenotype caused by VCP gene mutation in a Finnish family with nine affected members in three generations. Patients had onset of distal leg muscle weakness and atrophy in the anterior compartment muscles after age 35, which caused a foot drop at age 50. None of the siblings had scapular winging, proximal myopathy, cardiomyopathy or respiratory problems during long-term follow-up. Three distal myopathy patients developed rapidly progressive dementia, became bedridden and died of cachexia and pneumonia and VCP gene mutation P137L (c.410C>T) was then identified in the family. Late onset autosomal dominant distal myopathy with rimmed vacuolar muscle pathology was not sufficient for exact diagnosis in this family until late-occurring dementia provided the clue for molecular diagnosis. VCP needs to be considered in the differential diagnostic work-up in patients with distal myopathy phenotype. Copyright © 2011 Elsevier B.V. All rights reserved.

Splanchnic Vein Thrombosis in the Myeloproliferative Neoplasms.

PubMed

Akpan, Imo J; Stein, Brady Lee

2018-06-01

To review the epidemiology, diagnostic challenges, pathogenesis, and treatment strategies for patients with myeloproliferative neoplasm-associated splanchnic vein thrombosis. The epidemiology of myeloproliferative neoplasm-associated splanchnic vein thrombosis (MPN-SVT) has been well characterized. While typical MPN-associated thrombosis affects older patients and involves the arterial circulation, MPN-SVT mostly impacts younger women. An association with JAK2 V617F is well-known; recent studies have demonstrated only a weak association with CALR mutations. JAK inhibition may represent a novel treatment strategy, complementing anticoagulation, and management of portal hypertension. While the epidemiology has been well characterized, more work is needed to identify novel contributors to disease pathogenesis, beyond the JAK2 V617F mutation itself, and endothelial compromise. Testing for MPN mutations in the setting of non-cirrhotic SVT is commonplace; JAK2 V617F is the most likely to be identified. Testing for CALR or MPL mutations requires clinical judgement, though not unreasonable. The mainstay of therapy is indefinite anticoagulation; the role of direct oral anticoagulants is unclear. JAK inhibition may play a role in addressing associated splenomegaly and portal hypertension.

Genetic diversity in the interference selection limit.

PubMed

Good, Benjamin H; Walczak, Aleksandra M; Neher, Richard A; Desai, Michael M

2014-03-01

Pervasive natural selection can strongly influence observed patterns of genetic variation, but these effects remain poorly understood when multiple selected variants segregate in nearby regions of the genome. Classical population genetics fails to account for interference between linked mutations, which grows increasingly severe as the density of selected polymorphisms increases. Here, we describe a simple limit that emerges when interference is common, in which the fitness effects of individual mutations play a relatively minor role. Instead, similar to models of quantitative genetics, molecular evolution is determined by the variance in fitness within the population, defined over an effectively asexual segment of the genome (a "linkage block"). We exploit this insensitivity in a new "coarse-grained" coalescent framework, which approximates the effects of many weakly selected mutations with a smaller number of strongly selected mutations that create the same variance in fitness. This approximation generates accurate and efficient predictions for silent site variability when interference is common. However, these results suggest that there is reduced power to resolve individual selection pressures when interference is sufficiently widespread, since a broad range of parameters possess nearly identical patterns of silent site variability.

The detection of large deletions or duplications in genomic DNA.

PubMed

Armour, J A L; Barton, D E; Cockburn, D J; Taylor, G R

2002-11-01

While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications. Copyright 2002 Wiley-Liss, Inc.

Myosin storage myopathy associated with a heterozygous missense mutation in MYH7.

PubMed

Tajsharghi, Homa; Thornell, Lars-Eric; Lindberg, Christopher; Lindvall, Björn; Henriksson, Karl-Gösta; Oldfors, Anders

2003-10-01

Myosin constitutes the major part of the thick filaments in the contractile apparatus of striated muscle. MYH7 encodes the slow/beta-cardiac myosin heavy chain (MyHC), which is the main MyHC isoform in slow, oxidative, type 1 muscle fibers of skeletal muscle. It is also the major MyHC isoform of cardiac ventricles. Numerous missense mutations in the globular head of slow/beta-cardiac MyHC are associated with familial hypertrophic cardiomyopathy. We identified a missense mutation, Arg1845Trp, in the rod region of slow/beta-cardiac MyHC in patients with a skeletal myopathy from two different families. The myopathy was characterized by muscle weakness and wasting with onset in childhood and slow progression, but no overt cardiomyopathy. Slow, oxidative, type 1 muscle fibers showed large inclusions consisting of slow/beta-cardiac MyHC. The features were similar to a previously described entity: hyaline body myopathy. Our findings indicate that the mutated residue of slow/beta-cardiac MyHC is essential for the assembly of thick filaments in skeletal muscle. We propose the term myosin storage myopathy for this disease.

Six Highly Conserved Targets of RNAi Revealed in HIV-1-Infected Patients from Russia Are Also Present in Many HIV-1 Strains Worldwide.

PubMed

Kretova, Olga V; Fedoseeva, Daria M; Gorbacheva, Maria A; Gashnikova, Natalya M; Gashnikova, Maria P; Melnikova, Nataliya V; Chechetkin, Vladimir R; Kravatsky, Yuri V; Tchurikov, Nickolai A

2017-09-15

RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs) due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%-97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT), integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G) in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growth.

PubMed

Jeng, Jaan-Yeh; Yeh, Tien-Shun; Lee, Jing-Wen; Lin, Shyh-Hsiang; Fong, Tsorng-Han; Hsieh, Rong-Hong

2008-02-01

To examine whether a reduction in the mtDNA level will compromise mitochondrial biogenesis and mitochondrial function, we created a cell model with depleted mtDNA. Stable transfection of small interfering (si)RNA of mitochondrial transcription factor A (Tfam) was used to interfere with Tfam gene expression. Selected stable clones showed 60-95% reduction in Tfam gene expression and 50-90% reduction in cytochrome b (Cyt b) gene expression. Tfam gene knockdown clones also showed decreased mtDNA-encoded cytochrome c oxidase subunit I (COX I) protein expression. However, no significant differences in protein expression were observed in nuclear DNA (nDNA)-encoded mitochondrial respiratory enzyme subunits. The cell morphology changed from a rhombus-like to a spindle-like form as determined in clones with decreased expressions of Tfam, mtRNA, and mitochondrial proteins. The mitochondrial respiratory enzyme activities and ATP production in such clones were significantly lower. The proportions of mtDNA mutations including 8-hydroxy-2'-deoxyguanosine (8-OHdG), a 4,977-bp deletion, and a 3,243-point mutation were also examined in these clones. No obvious increase in mtDNA mutations was observed in mitochondrial dysfunctional cell clones. The mitochondrial respiratory activity and ATP production ability recovered in cells with increased mtDNA levels after removal of the specific siRNA treatment. These experimental results provide direct evidence to substantiate that downregulation of mtDNA copy number and expression may compromise mitochondrial function and subsequent cell growth and morphology. (c) 2007 Wiley-Liss, Inc.

Divergence times in Caenorhabditis and Drosophila inferred from direct estimates of the neutral mutation rate.

PubMed

Cutter, Asher D

2008-04-01

Accurate inference of the dates of common ancestry among species forms a central problem in understanding the evolutionary history of organisms. Molecular estimates of divergence time rely on the molecular evolutionary prediction that neutral mutations and substitutions occur at the same constant rate in genomes of related species. This underlies the notion of a molecular clock. Most implementations of this idea depend on paleontological calibration to infer dates of common ancestry, but taxa with poor fossil records must rely on external, potentially inappropriate, calibration with distantly related species. The classic biological models Caenorhabditis and Drosophila are examples of such problem taxa. Here, I illustrate internal calibration in these groups with direct estimates of the mutation rate from contemporary populations that are corrected for interfering effects of selection on the assumption of neutrality of substitutions. Divergence times are inferred among 6 species each of Caenorhabditis and Drosophila, based on thousands of orthologous groups of genes. I propose that the 2 closest known species of Caenorhabditis shared a common ancestor <24 MYA (Caenorhabditis briggsae and Caenorhabditis sp. 5) and that Caenorhabditis elegans diverged from its closest known relatives <30 MYA, assuming that these species pass through at least 6 generations per year; these estimates are much more recent than reported previously with molecular clock calibrations from non-nematode phyla. Dates inferred for the common ancestor of Drosophila melanogaster and Drosophila simulans are roughly concordant with previous studies. These revised dates have important implications for rates of genome evolution and the origin of self-fertilization in Caenorhabditis.

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

PubMed Central

Smith, Catherine E.; Mendillo, Marc L.; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S.; Desai, Arshad; Putnam, Christopher D.; Kolodner, Richard D.

2013-01-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway. PMID:24204293

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

PubMed

Smith, Catherine E; Mendillo, Marc L; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S; Desai, Arshad; Putnam, Christopher D; Kolodner, Richard D

2013-10-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Monotonicity of fitness landscapes and mutation rate control.

PubMed

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

2016-12-01

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

Novel GNE mutations in Italian families with autosomal recessive hereditary inclusion-body myopathy.

PubMed

Broccolini, Aldobrando; Ricci, Enzo; Cassandrini, Denise; Gliubizzi, Carla; Bruno, Claudio; Tonoli, Emmanuel; Silvestri, Gabriella; Pescatori, Mario; Rodolico, Carmelo; Sinicropi, Stefano; Servidei, Serenella; Zara, Federico; Minetti, Carlo; Tonali, Pietro A; Mirabella, Massimiliano

2004-06-01

The most common form of autosomal recessive (AR) hereditary inclusion-body myopathy (HIBM), originally described in Persian-Jewish families, is characterized by onset in early adult life with weakness and atrophy of distal lower limb muscles, which progress proximally and relatively spare the quadriceps. AR HIBM is associated with mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE) on chromosome 9p12-13. In the present study we have identified seven novel GNE mutations in patients from five unrelated Italian families with clinical and pathologic features indicative of AR HIBM. Four were missense mutations (c.1556A>G [p.N519S], c.79C>T [p.P27S], c.1798G>A [p.A600T] and c.616G>A [p.G206S]), two consisted in a single-base deletion (c.616delG [p.G206fsX4] and c.1130delT [p.I377fsX16]) and one in an intronic single-base insertion (c.1070+2dupT). These latter findings further extend the type of GNE mutations associated with HIBM. Furthermore, in one patient we also identified the c.737G>A [p.R246Q] missense mutation that corresponds to the one previously reported in a family from the Bahamas. Interestingly, in two of our families distinct mutations affected nucleotide c.616 in exon 3 (c.616delG and c.616G>A). The possibility of specific portions of the gene being more prone to mutations remains to be elucidated. Copyright 2004 Wiley-Liss, Inc.

Evolution of cooperation in a multidimensional phenotype space.

PubMed

Kroumi, Dhaker; Lessard, Sabin

2015-06-01

The emergence of cooperation in populations of selfish individuals is a fascinating topic that has inspired much theoretical work. An important model to study cooperation is the phenotypic model, where individuals are characterized by phenotypic properties that are visible to others. The phenotype of an individual can be represented for instance by a vector x = (x1,…,xn), where x1,…,xn are integers. The population can be well mixed in the sense that everyone is equally likely to interact with everyone else, but the behavioral strategies of the individuals can depend on their distance in the phenotype space. A cooperator can choose to help other individuals exhibiting the same phenotype and defects otherwise. Cooperation is said to be favored by selection if it is more abundant than defection in the stationary state. This means that the average frequency of cooperators in the stationary state strictly exceeds 1/2. Antal et al. (2009c) found conditions that ensure that cooperation is more abundant than defection in a one-dimensional (i.e. n = 1) and an infinite-dimensional (i.e. n = ∞) phenotype space in the case of the Prisoner's Dilemma under weak selection. However, reality lies between these two limit cases. In this paper, we derive the corresponding condition in the case of a phenotype space of any finite dimension. This is done by applying a perturbation method to study a mutation-selection equilibrium under weak selection. This condition is obtained in the limit of a large population size by using the ancestral process. The best scenario for cooperation to be more likely to evolve is found to be a high population-scaled phenotype mutation rate, a low population-scaled strategy mutation rate and a high phenotype space dimension. The biological intuition is that a high population-scaled phenotype mutation rate reduces the quantity of interactions between cooperators and defectors, while a high population-scaled strategy mutation rate introduces newly mutated defectors that invade groups of cooperators. Finally it is easier for cooperation to evolve in a phenotype space of higher dimension because it becomes more difficult for a defector to migrate to a group of cooperators. The difference is significant from n = 1 to n = 2 and from n = 2 to n = 3, but becomes small as soon as n ≥ 3. Copyright © 2015 Elsevier Inc. All rights reserved.

Novel autosomal dominant TNNT1 mutation causing nemaline myopathy.

PubMed

Konersman, Chamindra G; Freyermuth, Fernande; Winder, Thomas L; Lawlor, Michael W; Lagier-Tourenne, Clotilde; Patel, Shailendra B

2017-11-01

Nemaline myopathy (NEM) is one of the three major forms of congenital myopathy and is characterized by diffuse muscle weakness, hypotonia, respiratory insufficiency, and the presence of nemaline rod structures on muscle biopsy. Mutations in troponin T1 (TNNT1) is 1 of 10 genes known to cause NEM. To date, only homozygous nonsense mutations or compound heterozygous truncating or internal deletion mutations in TNNT1 gene have been identified in NEM. This extended family is of historical importance as some members were reported in the 1960s as initial evidence that NEM is a hereditary disorder. Proband and extended family underwent Sanger sequencing for TNNT1. We performed RT-PCR and immunoblot on muscle to assess TNNT1 RNA expression and protein levels in proband and father. We report a novel heterozygous missense mutation of TNNT1 c.311A>T (p.E104V) that segregated in an autosomal dominant fashion in a large family residing in the United States. Extensive sequencing of the other known genes for NEM failed to identify any other mutant alleles. Muscle biopsies revealed a characteristic pattern of nemaline rods and severe myofiber hypotrophy that was almost entirely restricted to the type 1 fiber population. This novel mutation alters a residue that is highly conserved among vertebrates. This report highlights not only a family with autosomal dominant inheritance of NEM, but that this novel mutation likely acts via a dominant negative mechanism. © 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Uncommon BRAF mutations in the follicular variant of thyroid papillary carcinoma: New insights.

PubMed

Rossi, Esther Diana; Martini, Maurizio; Bizzarro, Tommaso; Capodimonti, Sara; Cenci, Tonia; Lombardi, Celestino Pio; Pontecorvi, Alfredo; Fadda, Guido; Larocca, Luigi Maria

2015-10-01

Mutational analysis is reshaping the practice of fine-needle aspiration cytology for the diagnosis of thyroid nodules. The v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) valine (V) to glutamic acid (E) substitution at codon 600 (BRAF(V600E)) is the most effective diagnostic/prognostic marker and is used mainly for papillary thyroid carcinomas (PTCs). Although BRAF(V600E) represents 95% of all BRAF mutations, uncommon BRAF mutations have been identified in thyroid carcinomas. For the current study, the authors evaluated morphologic (plump pink cells and sickle-shaped nuclei) anti-BRAF(V600E) antibody (VE1) immunocytochemical and molecular findings of BRAF mutations in PTCs and in the follicular variant of PTC (FVPC). Between January 2013 and June 2014, there were 150 cytologic samples with surgical follow-up at the authors' institution. BRAF mutations, which were identified using liquid-based cytology, were classified into wild-type BRAF, BRAF(V600E), and uncommon BRAF mutations. All clinicopathologic correlations between BRAF and FVPCs were analyzed. Forty-four of 150 samples were identified as benign histologic lesions, and the authors focused on the 106 cytologic samples from patients who had malignant outcomes (60 PTCs and 46 FVPCs). The series included 16 follicular neoplasms, 36 samples diagnosed as suspicious of malignancy, and 54 samples diagnosed as positive for malignancy. The BRAF(V600E) mutation was detected in 17.4% of FVPCs and in 66.6% of PTCs, whereas uncommon BRAF mutations were detected only in FVPCs. Plump pink cells and VE1 expression were not identified in samples that had uncommon BRAF mutations. VE1 immunocytochemistry yielded positive results in all 36 samples that had the BRAF(V600E) mutation. Uncommon BRAF mutations were observed only in FVPCs and were linked to less aggressive behavior. Negative/weak VE1 expression was observed in both wild-type and uncommon BRAF mutations. The current investigation did not reveal any plump cells or morphologic BRAF findings in samples that had uncommon BRAF mutations. In the authors' experience, BRAF mutations detected by DNA methods were more accurate in identifying FVPCs. © 2015 American Cancer Society.

Reassessment of the putative role of BLK-p.A71T loss-of-function mutation in MODY and type 2 diabetes.

PubMed

Bonnefond, A; Yengo, L; Philippe, J; Dechaume, A; Ezzidi, I; Vaillant, E; Gjesing, A P; Andersson, E A; Czernichow, S; Hercberg, S; Hadjadj, S; Charpentier, G; Lantieri, O; Balkau, B; Marre, M; Pedersen, O; Hansen, T; Froguel, P; Vaxillaire, M

2013-03-01

MODY is believed to be caused by at least 13 different genes. Five rare mutations at the BLK locus, including only one non-synonymous p.A71T variant, were reported to segregate with diabetes in three MODY families. The p.A71T mutation was shown to abolish the enhancing effect of BLK on insulin content and secretion from pancreatic beta cell lines. Here, we reassessed the contribution of BLK to MODY and tested the effect of BLK-p.A71T on type 2 diabetes risk and variations in related traits. BLK was sequenced in 64 unelucidated MODY samples. The BLK-p.A71T variant was genotyped in a French type 2 diabetes case-control study including 4,901 cases and 4,280 controls, and in the DESIR (Data from an Epidemiological Study on the Insulin Resistance Syndrome) and SUVIMAX (Supplementation en Vitamines et Mineraux Antioxydants) population-based cohorts (n = 6,905). The variant effects were assessed by logistic and linear regression models. No rare non-synonymous BLK mutations were found in the MODY patients. The BLK p.A71T mutation was present in 52 normoglycaemic individuals, making it very unlikely that this loss-of-function mutation causes highly penetrant MODY. We found a nominal association between this variant and increased type 2 diabetes risk, with an enrichment of the mutation in the obese diabetic patients, although no significant association with BMI was identified. No mutation in BLK was found in our MODY cohort. From our findings, the BLK-p.A71T mutation may weakly influence type 2 diabetes risk in the context of obesity; however, this will require further validation.

X-linked Charcot-Marie-Tooth (CMT) neuropathies (CMTX1, CMTX2, CMTX3) show different clinical phenotype and molecular genetics

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

Ionasescu, V.V.; Searby, C.C.; Ionasescu, R.

1994-09-01

The purpose of this study was to compare the X-linked dominant type CMTX1 (20 families) with X-linked recessive types CMTX2 and CMTX3 (2 families). The clinical phenotype was consistent with CMT peripheral neuropathy in all cases including distal weakness, atrophy and sensory loss, pes cavus and areflexia. Additional clinicial involvement of the central nervous system was present in one family with CMTX2 (mental retardation) and one family with CMTX3 (spastic paraparesis). Tight genetic linkage to Xq13.1 was present in 20 families with CMTX1 (Z=34.07 at {theta}=0) for the marker DXS453. Fifteen of the CMTX1 families showed point mutations of themore » connexin 32 coding region (5 nonsense mutations, 8 missense mutations, 2 deletions). Five CMTX1 neuropathy families showed no evidence of point mutations of the CX32 coding sequence. These findings suggest that the CMTX1 neuropathy genotype in these families may be the result of promoter mutations, 3{prime}-untranslated region mutations or exon/intron splice site mutations or a mutation with a different type of connexin but which has close structural similarities to CX32. No mutations of the CX32 coding region were found in the CMTX2 or CMTX3 families. Linkage to Xq13.1 was excluded in both families. Genetic linkage to Xp22.2 was present in the CMTX2 family (Z=3.54 at {theta}=0) for the markers DXS987 and DXS999. Suggestion of linkage to Xq26 (Z=1.81 at {theta}=0) for the marker DXS86 was present in the CMTX3 family.« less

THYROID HORMONE RECEPTOR BETA GENE MUTATION (P453A) IN A TURKISH FAMILY PRODUCING RESISTANCE TO THYROID HORMONE

PubMed Central

Bayraktaroglu, Taner; Noel, Janet; Mukaddes, Nahit Motavalli; Refetoff, Samuel

2018-01-01

Two members of a Turkish family, a mother and son, had thyroid function tests suggestive of resistance to thyroid hormone (RTH). The clinical presentation was, however, different. The mother (proposita) had palpitation, weakness, tiredness, nervousness, dry mouth and was misdiagnosed as having multinodular toxic goiter which was treated with antithyroid drugs and partial thyroidectomy. Her younger son had attention deficit hyperactivity disorder and primary encopresis, but normal intellectual quotient. Both had elevated serum iodothyronine levels with nonsuppressed thyrotropin. A mutation in one allele of the thyroid hormone receptor beta gene (P453A) was identified, providing a genetic confirmation for the diagnosis of RTH. PMID:18561095

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes.

PubMed

Piluso, G; Politano, L; Aurino, S; Fanin, M; Ricci, E; Ventriglia, V M; Belsito, A; Totaro, A; Saccone, V; Topaloglu, H; Nascimbeni, A C; Fulizio, L; Broccolini, A; Canki-Klain, N; Comi, L I; Nigro, G; Angelini, C; Nigro, V

2005-09-01

The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. To obtain unbiased information on the consequences of CAPN3 mutations. 530 subjects with different grades of symptoms and 300 controls. High throughput denaturing HPLC analysis of DNA pools. 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. A non-invasive and cost-effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator.

PubMed

Lee, Sooncheol; Nguyen, Huong Minh; Kang, Changwon

2010-10-01

No biological function has been identified for tiny RNA transcripts that are abortively and repetitiously released from initiation complexes of RNA polymerase in vitro and in vivo to date. In this study, we show that abortive initiation affects termination in transcription of bacteriophage T7 gene 10. Specifically, abortive transcripts produced from promoter phi 10 exert trans-acting antitermination activity on terminator T phi both in vitro and in vivo. Following abortive initiation cycling of T7 RNA polymerase at phi 10, short G-rich and oligo(G) RNAs were produced and both specifically sequestered 5- and 6-nt C + U stretch sequences, consequently interfering with terminator hairpin formation. This antitermination activity depended on sequence-specific hybridization of abortive transcripts with the 5' but not 3' half of T phi RNA. Antitermination was abolished when T phi was mutated to lack a C + U stretch, but restored when abortive transcript sequence was additionally modified to complement the mutation in T phi, both in vitro and in vivo. Antitermination was enhanced in vivo when the abortive transcript concentration was increased via overproduction of RNA polymerase or ribonuclease deficiency. Accordingly, antitermination activity exerted on T phi by abortive transcripts should facilitate expression of T phi-downstream promoter-less genes 11 and 12 in T7 infection of Escherichia coli.

Production of Viable Gametes without Meiosis in Maize Deficient for an ARGONAUTE Protein[W

PubMed Central

Singh, Manjit; Goel, Shalendra; Meeley, Robert B.; Dantec, Christelle; Parrinello, Hugues; Michaud, Caroline; Leblanc, Olivier; Grimanelli, Daniel

2011-01-01

Apomixis is a form of asexual reproduction through seeds in angiosperms. Apomictic plants bypass meiosis and fertilization, developing offspring that are genetically identical to their mother. In a genetic screen for maize (Zea mays) mutants mimicking aspects of apomixis, we identified a dominant mutation resulting in the formation of functional unreduced gametes. The mutant shows defects in chromatin condensation during meiosis and subsequent failure to segregate chromosomes. The mutated locus codes for AGO104, a member of the ARGONAUTE family of proteins. AGO104 accumulates specifically in somatic cells surrounding the female meiocyte, suggesting a mobile signal rather than cell-autonomous control. AGO104 is necessary for non-CG methylation of centromeric and knob-repeat DNA. Digital gene expression tag profiling experiments using high-throughput sequencing show that AGO104 influences the transcription of many targets in the ovaries, with a strong effect on centromeric repeats. AGO104 is related to Arabidopsis thaliana AGO9, but while AGO9 acts to repress germ cell fate in somatic tissues, AGO104 acts to repress somatic fate in germ cells. Our findings show that female germ cell development in maize is dependent upon conserved small RNA pathways acting non-cell-autonomously in the ovule. Interfering with this repression leads to apomixis-like phenotypes in maize. PMID:21325139

Skeletal Characterization of the Fgfr3 Mouse Model of Achondroplasia Using Micro-CT and MRI Volumetric Imaging.

PubMed

Shazeeb, Mohammed Salman; Cox, Megan K; Gupta, Anurag; Tang, Wen; Singh, Kuldeep; Pryce, Cynthia T; Fogle, Robert; Mu, Ying; Weber, William D; Bangari, Dinesh S; Ying, Xiaoyou; Sabbagh, Yves

2018-01-11

Achondroplasia, the most common form of dwarfism, affects more than a quarter million people worldwide and remains an unmet medical need. Achondroplasia is caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene which results in over-activation of the receptor, interfering with normal skeletal development leading to disproportional short stature. Multiple mouse models have been generated to study achondroplasia. The characterization of these preclinical models has been primarily done with 2D measurements. In this study, we explored the transgenic model expressing mouse Fgfr3 containing the achondroplasia mutation G380R under the Col2 promoter (Ach). Survival and growth rate of the Ach mice were reduced compared to wild-type (WT) littermates. Axial skeletal defects and abnormalities of the sternebrae and vertebrae were observed in the Ach mice. Further evaluation of the Ach mouse model was performed by developing 3D parameters from micro-computed tomography (micro-CT) and magnetic resonance imaging (MRI). The 3-week-old mice showed greater differences between the Ach and WT groups compared to the 6-week-old mice for all parameters. Deeper understanding of skeletal abnormalities of this model will help guide future studies for evaluating novel and effective therapeutic approaches for the treatment of achondroplasia.

Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans

PubMed Central

Romeo, Stefano; Yin, Wu; Kozlitina, Julia; Pennacchio, Len A.; Boerwinkle, Eric; Hobbs, Helen H.; Cohen, Jonathan C.

2008-01-01

The relative activity of lipoprotein lipase (LPL) in different tissues controls the partitioning of lipoprotein-derived fatty acids between sites of fat storage (adipose tissue) and oxidation (heart and skeletal muscle). Here we used a reverse genetic strategy to test the hypothesis that 4 angiopoietin-like proteins (ANGPTL3, -4, -5, and -6) play key roles in triglyceride (TG) metabolism in humans. We re-sequenced the coding regions of the genes encoding these proteins and identified multiple rare nonsynonymous (NS) sequence variations that were associated with low plasma TG levels but not with other metabolic phenotypes. Functional studies revealed that all mutant alleles of ANGPTL3 and ANGPTL4 that were associated with low plasma TG levels interfered either with the synthesis or secretion of the protein or with the ability of the ANGPTL protein to inhibit LPL. A total of 1% of the Dallas Heart Study population and 4% of those participants with a plasma TG in the lowest quartile had a rare loss-of-function mutation in ANGPTL3, ANGPTL4, or ANGPTL5. Thus, ANGPTL3, ANGPTL4, and ANGPTL5, but not ANGPTL6, play nonredundant roles in TG metabolism, and multiple alleles at these loci cumulatively contribute to variability in plasma TG levels in humans. PMID:19075393

Proprotein convertase subtilisin/kexin type 9 (PCSK9): from structure-function relation to therapeutic inhibition.

PubMed

Tibolla, G; Norata, G D; Artali, R; Meneghetti, F; Catapano, A L

2011-11-01

This short review aims at summarizing the current information on Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) structure and function focusing also on the therapeutic possibilities based on the inhibition of this protein. PCSK9 has been recently discovered as the third gene involved in autosomal dominant hypercholesterolemia. PCSK9 binds and favors degradation of the low-density lipoprotein receptor (LDLR) and thereby modulates the plasma levels of LDL-cholesterol (LDL-C). Some of the natural occurring PCSK9 mutations increase the protein function (gain of function) and cause hypercholesterolemia, whereas loss of function mutations associate with hypocholesterolemia. Since the loss of a functional PCSK9 in humans is not associated with apparent deleterious effects, this protease is an attractive target for the development of lowering plasma LDL-C agents, either alone or in combination with statins. Inhibition of PCSK9 is emerging as a novel strategy for the treatment of hypercholesterolemia and data obtained from pre-clinical studies show that use of monoclonal antibodies, antisense oligonucleotides and short interfering RNA are effective in reducing LDL-C, clinical studies, accompanied by a better understanding of PCSK9 biology, are now necessary to address whether these new compounds will have a future in clinical practice. Copyright © 2011 Elsevier B.V. All rights reserved.

A Mutation Affecting the Sodium/Proton Exchanger, "SLC9A6," Causes Mental Retardation with Tau Deposition

ERIC Educational Resources Information Center

Garbern, James Y.; Neumann, Manuela; Trojanowski, John Q.; Lee, Virginia M.-Y.; Feldman, Gerald; Norris, Joy W.; Friez, Michael J.; Schwartz, Charles E.; Stevenson, Roger; Sima, Anders A. F.

2010-01-01

We have studied a family with severe mental retardation characterized by the virtual absence of speech, autism spectrum disorder, epilepsy, late-onset ataxia, weakness and dystonia. Post-mortem examination of two males revealed widespread neuronal loss, with the most striking finding being neuronal and glial tau deposition in a pattern reminiscent…

Glutaric aciduria type 2, late onset type in Thai siblings with myopathy.

PubMed

Wasant, Pornswan; Kuptanon, Chulaluck; Vattanavicharn, Nithiwat; Liammongkolkul, Somporn; Ratanarak, Pisanu; Sangruchi, Tumtip; Yamaguchi, Seiji

2010-10-01

Reported here is a novel presentation of late onset glutaric aciduria type 2 in two Thai siblings. A 9-year-old boy presented with gradual onset of proximal muscle weakness for 6 weeks. The initial diagnosis was postviral myositis, and then polymyositis. Electromyography and nerve conduction velocity testing indicated a myopathic pattern. Muscle biopsy revealed excessive accumulation of fat. Acylcarnitine profiling led to the diagnosis of glutaric aciduria type 2. Immunoblot analysis of electron-transferring-flavoprotein and its dehydrogenase electron-transferring-flavoprotein dehydrogenase led to mutation analysis of the ETFDH gene, which revealed two different pathogenic mutations in both alleles and confirmed the diagnosis of glutaric aciduria type 2 caused by electron-transferring-flavoprotein dehydrogenase deficiency. The boy recovered completely after treatment. Later, his younger sibling became symptomatic; the same diagnosis was confirmed, and treatment was similarly effective. Acylcarnitine profiling was a crucial investigation in making this diagnosis in the presence of normal urine organic acid findings. Late onset glutaric aciduria type 2, a rare cause of muscle weakness in children, should be included in the differential diagnosis of myopathy. Copyright © 2010 Elsevier Inc. All rights reserved.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.

Predicted Mutation Strength of Nontruncating PKD1 Mutations Aids Genotype-Phenotype Correlations in Autosomal Dominant Polycystic Kidney Disease.

PubMed

Heyer, Christina M; Sundsbak, Jamie L; Abebe, Kaleab Z; Chapman, Arlene B; Torres, Vicente E; Grantham, Jared J; Bae, Kyongtae T; Schrier, Robert W; Perrone, Ronald D; Braun, William E; Steinman, Theodore I; Mrug, Michal; Yu, Alan S L; Brosnahan, Godela; Hopp, Katharina; Irazabal, Maria V; Bennett, William M; Flessner, Michael F; Moore, Charity G; Landsittel, Douglas; Harris, Peter C

2016-09-01

Autosomal dominant polycystic kidney disease (ADPKD) often results in ESRD but with a highly variable course. Mutations to PKD1 or PKD2 cause ADPKD; both loci have high levels of allelic heterogeneity. We evaluated genotype-phenotype correlations in 1119 patients (945 families) from the HALT Progression of PKD Study and the Consortium of Radiologic Imaging Study of PKD Study. The population was defined as: 77.7% PKD1, 14.7% PKD2, and 7.6% with no mutation detected (NMD). Phenotypic end points were sex, eGFR, height-adjusted total kidney volume (htTKV), and liver cyst volume. Analysis of the eGFR and htTKV measures showed that the PKD1 group had more severe disease than the PKD2 group, whereas the NMD group had a PKD2-like phenotype. In both the PKD1 and PKD2 populations, men had more severe renal disease, but women had larger liver cyst volumes. Compared with nontruncating PKD1 mutations, truncating PKD1 mutations associated with lower eGFR, but the mutation groups were not differentiated by htTKV. PKD1 nontruncating mutations were evaluated for conservation and chemical change and subdivided into strong (mutation strength group 2 [MSG2]) and weak (MSG3) mutation groups. Analysis of eGFR and htTKV measures showed that patients with MSG3 but not MSG2 mutations had significantly milder disease than patients with truncating cases (MSG1), an association especially evident in extreme decile populations. Overall, we have quantified the contribution of genic and PKD1 allelic effects and sex to the ADPKD phenotype. Intrafamilial correlation analysis showed that other factors shared by families influence htTKV, with these additional genetic/environmental factors significantly affecting the ADPKD phenotype. Copyright © 2016 by the American Society of Nephrology.

Recognition Mechanism of siRNA by Viral p19 Suppressor of RNA Silencing: A Molecular Dynamics Study

PubMed Central

Xia, Zhen; Zhu, Zhihong; Zhu, Jun; Zhou, Ruhong

2009-01-01

The p19 protein (p19) encoded from Tombusvirus is involved in various activities such as pathogenicity and virus transport. Recent studies have found that p19 is a plant suppressor of RNA silencing, which binds to short interfering RNAs (siRNAs) with high affinity. We use molecular dynamics (MD) simulations of the wild-type and mutant p19 protein (W39 and W42G) binding with a 21-nt siRNA duplex to study the p19-siRNA recognition mechanism and mutation effects. Our simulations with standard MD and steered molecular dynamics have shown that the double mutant structure is indeed much less stable than the wild-type, consistent with the recent experimental findings. Comprehensive structural analysis also shows that the W39/42G mutations first induce the loss of stacking interactions between p19 and siRNA, Trp42-Cyt1 (Cyt1 from the 5′ to 3′ strand) and Trp39-Gua′19 (Gua19 from the 3′ to 5′ strand), and then breaks the hydrophobic core formed by W39-W42 with nucleotide basepairs in the wild-type. The steered molecular dynamics simulations also show that the mutant p19 complex is “decompounded” very fast under a constant separation force, whereas the wild-type remains largely intact under the same steering force. Moreover, we have used the free energy perturbation to predict a binding affinity loss of 6.98 ± 0.95 kcal/mol for the single mutation W39G, and 12.8 ± 1.0 kcal/mol loss for the double mutation W39/42G, with the van der Waals interactions dominating the contribution (∼90%). These results indicate that the W39/42G mutations essentially destroy the important p19-siRNA recognition by breaking the strong stacking interaction between Cyt1 and Gua′19 with end-capping tryptophans. These large scale simulations might provide new insights to the interactions and co-evolution relationship between RNA virus proteins and their hosts. PMID:19254536

Impact of epidermal growth factor receptor gene expression level on clinical outcomes in epidermal growth factor receptor mutant lung adenocarcinoma patients taking first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

PubMed

Chang, Huang-Chih; Chen, Yu-Mu; Tseng, Chia-Cheng; Huang, Kuo-Tung; Wang, Chin-Chou; Chen, Yung-Che; Lai, Chien-Hao; Fang, Wen-Feng; Kao, Hsu-Ching; Lin, Meng-Chih

2017-03-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are first-choice treatments for advanced non-small-cell lung cancer patients harboring EGFR mutations. Although EGFR mutations are strongly predictive of patients' outcomes and their response to treatment with EGFR-TKIs, early failure of first-line therapy with EGFR-TKIs in patients with EGFR mutations is not rare. Besides several clinical factors influencing EGFR-TKI efficacies studied earlier such as the Eastern Cooperative Oncology Group performance status or uncommon mutation, we would like to see whether semi-quantify EGFR mutation gene expression calculated by 2 -ΔΔct was a prognostic factor in EGFR-mutant non-small cell lung cancer patients receiving first-line EGFR-TKIs. This retrospective study reviews 926 lung cancer patients diagnosed from January 2011 to October 2013 at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Of 224 EGFR-mutant adenocarcinoma patients, 148 patients who had 2 -ΔΔct data were included. The best cutoff values of 2 -ΔΔct for in-frame deletions in exon 19 (19 deletion) and a position 858 substituted from leucine (L) to an arginine (R) in exon 21 (L858R) were determined using receiver operating characteristic curves. Patients were divided into high and low 2 -ΔΔct expression based on the above cutoff level. The best cutoff point of 2 -ΔΔct value of 19 deletion and L858R was 31.1 and 104.7, respectively. In all, 92 (62.1%) patients showed high 2 -ΔΔct expression and 56 patients (37.9%) low 2 -ΔΔct expression. The mean age was 65.6 years. Progression-free survival of 19 deletion mutant patients with low versus high expression level was 17.07 versus 12.04 months (P = 0.004), respectively. Progression-free survival of L858 mutant patients was 13.75 and 7.96 months (P = 0.008), respectively. EGFR-mutant lung adenocarcinoma patients with lower EGFR gene expression had longer progression-free survival duration without interfering overall survival.

Lung Cancer: One Disease or Many.

PubMed

O'Brien, Timothy D; Jia, Peilin; Aldrich, Melinda C; Zhao, Zhongming

2018-06-05

Lung cancer is classified as a single entity comprised of multiple histological subtypes. But how similar are these subtypes on a genetic level? This paper aims to address this question through a concise overview of germline and somatic differences between small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma. We reveal the weak overlap found between these 3 lung cancer subtypes using published data from one of the largest germline genetic studies on lung cancer to date and somatic mutation data from Catalogue of Somatic Mutations in Cancer (COSMIC). These data indicate that these 3 subtypes share very little with each other at the genetic level. At the germline SNP level, only 24 independent SNPs from 2 chromosomes were shared across all 3 subtypes. We also demonstrate that only 30 unique cancer-specific mutations overlap the 3 subtypes from COSMIC, and that this is fewer than overlapping mutations chosen at random. Finally, we show that only 3 somatic mutational signatures are shared between these 3 subtypes. This paper highlights that these 3 lung cancer subtypes may be distinct diseases at the genetic level. In the era of precision medicine, we feel that these genomic differences will be of utmost importance in the choice of lung cancer therapy in the future. © 2018 S. Karger AG, Basel.

MITOCHONDRIAL DNA DEPLETION SYNDROME DUE TO MUTATIONS IN THE RRM2B GENE

PubMed Central

Bornstein, Belén; Area, Estela; Flanigan, Kevin M.; Ganesh, Jaya; Jayakar, Parul; Swoboda, Kathryn J.; Coku, Jorida; Naini, Ali; Shanske, Sara; Tanji, Kurenai; Hirano, Michio; DiMauro, Salvatore

2014-01-01

Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in The RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in 7 infants from 4 families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at three months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exon 6, 8 and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy. PMID:18504129

Mitochondrial DNA depletion syndrome due to mutations in the RRM2B gene.

PubMed

Bornstein, Belén; Area, Estela; Flanigan, Kevin M; Ganesh, Jaya; Jayakar, Parul; Swoboda, Kathryn J; Coku, Jorida; Naini, Ali; Shanske, Sara; Tanji, Kurenai; Hirano, Michio; DiMauro, Salvatore

2008-06-01

Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in the RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in seven infants from four families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at 3 months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exons 6, 8, and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy.

Mutations in eukaryotic release factors 1 and 3 act as general nonsense suppressors in Drosophila.

PubMed Central

Chao, Anna T; Dierick, Herman A; Addy, Tracie M; Bejsovec, Amy

2003-01-01

In a screen for suppressors of the Drosophila wingless(PE4) nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens. PMID:14573473

High frequency of mutations in codon 98 of the peripheral myelin protein Po gene in 20 French CMT1 patients

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

Rougher, H.; LeGuern, E. Gouider, R.

1996-03-01

Charcot-Marie-Tooth disease, characterized by distal muscle weakness and amyotrophy, decreased or absent tendon reflexes, and high arched feet, is the most common inherited peripheral neuropathy, with a prevalence of 1 in 2,500. Two types of CMT have been distinguished on the basis of nerve conduction velocities. CMT type 1 is the most frequent, with markedly slowed velocities ({<=}40 m/s) associated with hypertrophic onion bulb changes on nerve biopsy. Autosomal dominant CMT1 is genetically heterogeneous: CMT1A is caused by a 1.5-Mb duplication in 17p11.2 and, more rarely, by a point mutation in tha PMP22 (peripheral myelin protein, 22 kD) gene locatedmore » in the duplicated region; CMT1B results from mutations in the Po (peripheral myelin protein zero) gene in 1q22-23. Forty-five percent (7/16) of the published mutations associated with CMT1 occur in exon 3 of Po. In order to determine the cause of CMT1 in 20 unrelated patients without 17p11.2 duplications, mutations were sought in exon 3 of Po with three techniques: nonradioactive SSCP, automated sequencing, and PCR enzymatic restriction. 18 refs., 2 figs.« less

The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway.

PubMed

Vasilcanu, Daiana; Girnita, Ada; Girnita, Leonard; Vasilcanu, Radu; Axelson, Magnus; Larsson, Olle

2004-10-14

The insulin-like growth factor-1 receptor (IGF-1R) is crucial for many functions in neoplastic cells, for example, antiapoptosis. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. PPP preferentially reduced phosphorylated Akt, as compared to phosphorylated Erk1/2, and caused apoptosis. Now, we aimed to investigate how PPP inhibits the IGF-1R tyrosine kinase (IGF-1RTK) and the PI3K/Akt apoptotic pathway. Using a baculovirus driven IGF-1RTK we found that PPP interfered with tyrosine phosphorylation in the activation loop of the kinase domain. Specifically, it blocked phosphorylation of tyrosine (Y) 1136, while sparing the two others (Y1131 and Y1135). To explore the impact of inhibition of Y1136 on Akt phosphorylation we transfected P6 cells (overexpressing IGF-1R) and malignant melanoma cells with different IGF-1R mutants, including Y1136F (tyrosine replaced by phenylalanine). Y1136F was found to strongly decrease IGF-1 stimulated phosphorylation of Akt. Conversely, Akt phosphorylation was weakly affected in the Y1131F transfectant. Taken together, our data suggest that the preferential inhibition of phosphorylated Akt, after PPP treatment, may be due to specific inhibition of Y1136. PPP was proven not to interfere directly with Akt or any of its downstream molecules in the apoptotic pathway.

Development of a Technique for Separating Raman Scattering Signals from Background Emission with Single-Shot Measurement Potential

NASA Technical Reports Server (NTRS)

Hartfield, Roy J., Jr.; Dobson, Chris; Eskridge, Richard; Wehrmeyer, Joseph A.

1997-01-01

A novel technique for extracting Q-branch Raman signals scattered by a diatomic species from the emission spectrum resulting from the irradiation of combustion products using a broadband excimer laser has been developed. This technique is based on the polarization characteristics of vibrational Raman scattering and can be used for both single-shot Raman extraction and time-averaged data collection. The Q-branch Raman signal has a unique set of polarization characteristics which depend on the direction of the scattering while fluorescence signals are unpolarized. For the present work, a calcite crystal is used to separate the horizonal component of a collected signal from the vertical component. The two components are then sent through a UV spectrometer and imaged onto an intensified CCD camera separately. The vertical component contains both the Raman signal and the interfering fluorescence signal. The horizontal component contains the fluorescence signal and a very weak component of the Raman signal; hence, the Raman scatter can be extracted by taking the difference between the two signals. The separation of the Raman scatter from interfering fluorescence signals is critically important to the interpretation of the Raman for cases in which a broadband ultraviolet (UV) laser is used as an excitation source in a hydrogen-oxygen flame and in all hydrocarbon flames. The present work provides a demonstration of the separation of the Raman scatter from the fluorescence background in real time.

Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

PubMed

Sundaramoorthy, Vinod; Walker, Adam K; Tan, Vanessa; Fifita, Jennifer A; Mccann, Emily P; Williams, Kelly L; Blair, Ian P; Guillemin, Gilles J; Farg, Manal A; Atkin, Julie D

2015-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that optineurin-myosin VI dysfunction is a common feature of both sporadic and familial ALS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of NCAM helps identify unusual phenotypes of hereditary inclusion-body myopathy.

PubMed

Broccolini, A; Gidaro, T; Tasca, G; Morosetti, R; Rodolico, C; Ricci, E; Mirabella, M

2010-07-20

Hereditary inclusion-body myopathy or distal myopathy with rimmed vacuoles (h-IBM/DMRV) is due to mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which codes for an enzyme of the sialic acid biosynthetic pathway. By Western blot (WB) analysis, we have previously shown that in h-IBM/DMRV muscle, the neural cell adhesion molecule (NCAM) has increased electrophoretic mobility that reflects reduced sialylation of the protein. To identify patients with h-IBM/DMRV with atypical clinical or pathologic phenotype using NCAM analysis and the possible cellular mechanism associated with the overall abnormal sialylation of NCAM observed in this disorder. WB analysis of NCAM was performed on muscle biopsies of 84 patients with an uncharacterized muscle disorder who were divided in the following 2 groups: 1) 46 patients with a proximal muscle weakness in whom the main limb-girdle muscular dystrophy syndromes had been ruled out; and 2) 38 patients with a distal distribution of weakness in whom a neurogenic affection had been excluded. Patients in whom a reduced sialylation of NCAM was suspected were studied for the presence of GNE mutations. In 3 patients, we found that NCAM had increased electrophoretic mobility, thus suggesting an abnormal sialylation of the protein. The genetic study demonstrated that they all carried pathogenic GNE mutations. Further studies demonstrated that hyposialylated NCAM, showing increased electrophoretic mobility on WB, is expressed by nonregenerating fibers in h-IBM/DMRV muscle. WB analysis of NCAM may be instrumental in the identification of h-IBM/DMRV with atypical clinical or pathologic features.

Population genetics inference for longitudinally-sampled mutants under strong selection.

PubMed

Lacerda, Miguel; Seoighe, Cathal

2014-11-01

Longitudinal allele frequency data are becoming increasingly prevalent. Such samples permit statistical inference of the population genetics parameters that influence the fate of mutant variants. To infer these parameters by maximum likelihood, the mutant frequency is often assumed to evolve according to the Wright-Fisher model. For computational reasons, this discrete model is commonly approximated by a diffusion process that requires the assumption that the forces of natural selection and mutation are weak. This assumption is not always appropriate. For example, mutations that impart drug resistance in pathogens may evolve under strong selective pressure. Here, we present an alternative approximation to the mutant-frequency distribution that does not make any assumptions about the magnitude of selection or mutation and is much more computationally efficient than the standard diffusion approximation. Simulation studies are used to compare the performance of our method to that of the Wright-Fisher and Gaussian diffusion approximations. For large populations, our method is found to provide a much better approximation to the mutant-frequency distribution when selection is strong, while all three methods perform comparably when selection is weak. Importantly, maximum-likelihood estimates of the selection coefficient are severely attenuated when selection is strong under the two diffusion models, but not when our method is used. This is further demonstrated with an application to mutant-frequency data from an experimental study of bacteriophage evolution. We therefore recommend our method for estimating the selection coefficient when the effective population size is too large to utilize the discrete Wright-Fisher model. Copyright © 2014 by the Genetics Society of America.

High-field asymmetric waveform ion mobility spectrometry for mass spectrometry-based proteomics.

PubMed

Swearingen, Kristian E; Moritz, Robert L

2012-10-01

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve the detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, the authors review recent developments in LC-FAIMS-MS and its application to MS-based proteomics.

Encapsidated Atom-Transfer Radical Polymerization in Qβ Virus-like Nanoparticles

PubMed Central

2015-01-01

Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qβ VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA. PMID:25073013

Chelation in root canal therapy reconsidered.

PubMed

Zehnder, Matthias; Schmidlin, Patrick; Sener, Beatrice; Waltimo, Tuomas

2005-11-01

The aim of this study was to assess interactions of EDTA and citric acid (CA) with sodium hypochlorite (NaOCl), the indispensable endodontic irrigant. Other chelators were simultaneously evaluated as possible alternatives: sodium triphosphate (STP), amino tris methylenephosphonic acid (ATMA), and 1- hydroxyethylidene-1, 1-bisphosphonate (HEBP). Available chlorine was titrated in chelator-NaOCl solutions. All chelators other than HEBP and STP caused an almost complete, immediate loss of available chlorine in solution. Atomic absorbtion spectrometry and SEM evaluation of root canal walls of instrumented teeth indicated that NaOCl had no negative effect on calcium-complexing ability of chelators. STP was too weak a complexing agent to warrant further studies. Finally, CA-, EDTA-, and HEBP-NaOCl mixtures were evaluated for their antimicrobial capacity. Again, EDTA and CA negatively interfered with NaOCl, while HEBP did not.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica.

PubMed

Maluf, Mirian Perez; da Silva, Carla Cristina; de Oliveira, Michelle de Paula Abreu; Tavares, Aline Gomes; Silvarolla, Maria Bernadete; Guerreiro, Oliveiro

2009-10-01

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

PubMed Central

2009-01-01

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence. PMID:21637458

Consequences of a novel caveolin-3 mutation in a large German family.

PubMed

Fischer, Dirk; Schroers, Anja; Blümcke, Ingmar; Urbach, Horst; Zerres, Klaus; Mortier, Wilhelm; Vorgerd, Matthias; Schröder, Rolf

2003-02-01

Mutations in the human caveolin-3 gene (cav-3) on chromosome 3p25 have been described in limb girdle muscular dystrophy, rippling muscle disease, hyperCKemia, and distal myopathy. Here, we describe the genetic, myopathological, and clinical findings in a large German family harboring a novel heterozygous mutation (GAC-->GAA) in codon 27 of the cav-3 gene. This missense mutation causes an amino acid change from asparagine to glutamate (Asp27Glu) in the N-terminal region of the Cav-3 protein, which leads to a drastic decrease of Cav-3 protein expression in skeletal muscle tissue. In keeping with an autosomal dominant mode of inheritance, this novel cav-3 mutation was found to cosegregate with neuromuscular involvement in the reported family. Ultrastructural analysis of Cav-3-deficient muscle showed an abnormal folding of the plasma membrane as well as multiple vesicular structures in the subsarcolemmal region. Neurological examination of all nine subjects from three generations harboring the novel cav-3 mutation showed clear evidence of rippling muscle disease. However, only two of these nine patients showed isolated signs of rippling muscle disease without muscle weakness or atrophy, whereas five had additional signs of a distal myopathy and two fulfilled the diagnostic criteria of a coexisting limb girdle muscular dystrophy. These findings indicate that mutations in the human cav-3 gene can lead to different and overlapping clinical phenotypes even within the same family. Different clinical phenotypes in caveolinopathies may be attributed to so far unidentified modifying factors/genes in the individual genetic background of affected patients.

Increased Oxidative Stress Markers in Cerebrospinal Fluid from Healthy Subjects with Parkinson's Disease-Associated LRRK2 Gene Mutations.

PubMed

Loeffler, David A; Klaver, Andrea C; Coffey, Mary P; Aasly, Jan O; LeWitt, Peter A

2017-01-01

Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene are the most frequent cause of inherited Parkinson's disease (PD). The most common PD-associated LRRK2 mutation, G2019S, induces increased production of reactive oxygen species in vitro . We therefore hypothesized that individuals with PD-associated LRRK2 mutations might have increased concentrations of oxidative stress markers and/or decreased total antioxidant capacity (TAC) in their cerebrospinal fluid (CSF). We measured two oxidative stress markers, namely 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-isoprostane (8-ISO), and TAC in CSF from LRRK2 mutation-bearing PD patients ( LRRK2 PD = 19), sporadic PD patients (sPD = 31), and healthy control subjects with or without these mutations ( LRRK2 CTL = 30, CTL = 27). 8-OHdG and 8-ISO levels were increased in LRRK2 CTL subjects, while TAC was similar between groups. 8-ISO was negatively correlated, and TAC was positively correlated, with Montreal Cognitive Assessment scores in LRRK2 PD, LRRK2 CTL, and CTL subjects. Correlations in both groups of PD patients between the two oxidative stress markers and Unified Parkinson Disease Rating Scale Total scores were weak, while TAC was negatively correlated with these scores. These findings suggest that oxidative stress may be increased in the CNS in healthy individuals with PD-associated LRRK2 mutations. Further, TAC may decrease in the CNS with the progression of PD, and when cognitive impairment is present regardless of the presence or absence of PD.

Increased Oxidative Stress Markers in Cerebrospinal Fluid from Healthy Subjects with Parkinson’s Disease-Associated LRRK2 Gene Mutations

PubMed Central

Loeffler, David A.; Klaver, Andrea C.; Coffey, Mary P.; Aasly, Jan O.; LeWitt, Peter A.

2017-01-01

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent cause of inherited Parkinson’s disease (PD). The most common PD-associated LRRK2 mutation, G2019S, induces increased production of reactive oxygen species in vitro. We therefore hypothesized that individuals with PD-associated LRRK2 mutations might have increased concentrations of oxidative stress markers and/or decreased total antioxidant capacity (TAC) in their cerebrospinal fluid (CSF). We measured two oxidative stress markers, namely 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-isoprostane (8-ISO), and TAC in CSF from LRRK2 mutation-bearing PD patients (LRRK2 PD = 19), sporadic PD patients (sPD = 31), and healthy control subjects with or without these mutations (LRRK2 CTL = 30, CTL = 27). 8-OHdG and 8-ISO levels were increased in LRRK2 CTL subjects, while TAC was similar between groups. 8-ISO was negatively correlated, and TAC was positively correlated, with Montreal Cognitive Assessment scores in LRRK2 PD, LRRK2 CTL, and CTL subjects. Correlations in both groups of PD patients between the two oxidative stress markers and Unified Parkinson Disease Rating Scale Total scores were weak, while TAC was negatively correlated with these scores. These findings suggest that oxidative stress may be increased in the CNS in healthy individuals with PD-associated LRRK2 mutations. Further, TAC may decrease in the CNS with the progression of PD, and when cognitive impairment is present regardless of the presence or absence of PD. PMID:28420983

Exome sequencing identifies titin mutations causing hereditary myopathy with early respiratory failure (HMERF) in families of diverse ethnic origins.

PubMed

Toro, Camilo; Olivé, Montse; Dalakas, Marinos C; Sivakumar, Kumaraswami; Bilbao, Juan M; Tyndel, Felix; Vidal, Noemí; Farrero, Eva; Sambuughin, Nyamkhishig; Goldfarb, Lev G

2013-03-20

Hereditary myopathy with early respiratory failure (HMERF) was described in several North European families and recently linked to a titin gene (TTN) mutation. We independently studied HMERF-like diseases with the purpose to identify the cause, refine diagnostic criteria, and estimate the frequency of this disease among myopathy patients of various ethnic origins. Whole exome sequencing analysis was carried out in a large U.S. family that included seven members suffering from skeletal muscle weakness and respiratory failure. Subsequent mutation screening was performed in further 45 unrelated probands with similar phenotypes. Studies included muscle strength evaluation, nerve conduction studies and concentric needle EMG, respiratory function test, cardiologic examination, and muscle biopsy. A novel TTN p.Gly30150Asp mutation was identified in the highly conserved A-band of titin that co-segregated with the disease in the U.S. family. Screening of 45 probands initially diagnosed as myofibrillar myopathy (MFM) but excluded based on molecular screening for the known MFM genes led to the identification of a previously reported TTN p.Cys30071Arg mutation in one patient. This same mutation was also identified in a patient with suspected HMERF. The p.Gly30150Asp and p.Cys30071Arg mutations are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin. Missense mutations in TTN are the cause of HMERF in families of diverse origins. A comparison of phenotypic features of HMERF caused by the three known TTN mutations in various populations allowed to emphasize distinct clinical/pathological features that can serve as the basis for diagnosis. The newly identified p.Gly30150Asp and the p.Cys30071Arg mutation are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.

Novel and general approach to linear filter design for contrast-to-noise ratio enhancement of magnetic resonance images with multiple interfering features in the scene

NASA Astrophysics Data System (ADS)

Soltanian-Zadeh, Hamid; Windham, Joe P.

1992-04-01

Maximizing the minimum absolute contrast-to-noise ratios (CNRs) between a desired feature and multiple interfering processes, by linear combination of images in a magnetic resonance imaging (MRI) scene sequence, is attractive for MRI analysis and interpretation. A general formulation of the problem is presented, along with a novel solution utilizing the simple and numerically stable method of Gram-Schmidt orthogonalization. We derive explicit solutions for the case of two interfering features first, then for three interfering features, and, finally, using a typical example, for an arbitrary number of interfering feature. For the case of two interfering features, we also provide simplified analytical expressions for the signal-to-noise ratios (SNRs) and CNRs of the filtered images. The technique is demonstrated through its applications to simulated and acquired MRI scene sequences of a human brain with a cerebral infarction. For these applications, a 50 to 100% improvement for the smallest absolute CNR is obtained.

Juvenile-onset Sporadic Amyotrophic Lateral Sclerosis with a Frameshift FUS Gene Mutation Presenting Unique Neuroradiological Findings and Cognitive Impairment.

PubMed

Hirayanagi, Kimitoshi; Sato, Masayuki; Furuta, Natsumi; Makioka, Kouki; Ikeda, Yoshio

2016-01-01

A 24-year-old Japanese woman developed anterocollis, weakness of the proximal arms, and subsequent cognitive impairment. A neurological examination revealed amyotrophic lateral sclerosis (ALS) without a family history. Systemic muscle atrophy progressed rapidly. Cerebral MRI clearly exhibited high signal intensities along the bilateral pyramidal tracts. An analysis of the FUS gene revealed a heterozygous two-base pair deletion, c.1507-1508delAG (p.G504WfsX515). A subset of juvenile-onset familial/sporadic ALS cases with FUS gene mutations reportedly demonstrates mental retardation or learning difficulty. Our study emphasizes the importance of conducting a FUS gene analysis in juvenile-onset ALS cases, even when no family occurrence is confirmed.

An Italian case of hereditary myopathy with early respiratory failure (HMERF) not associated with the titin kinase domain R279W mutation.

PubMed

Tasca, Giorgio; Mirabella, Massimiliano; Broccolini, Aldobrando; Monforte, Mauro; Sabatelli, Mario; Biscione, Gian Luca; Piluso, Giulio; Gualandi, Francesca; Tonali, Pietro Attilio; Udd, Bjarne; Ricci, Enzo

2010-11-01

Hereditary myopathy with early respiratory failure (HMERF) is a rare disorder characterized by severe respiratory involvement at onset, muscle weakness starting in the early adulthood, and cytoplasmic bodies with peculiar immunohistochemical reactivity on muscle biopsy. Here we describe a patient who presented with hypercapnic coma at age 32. A detailed light and electron microscopy analysis on muscle biopsy was performed and, together with clinical data, led to the diagnosis. The R279W mutation in the TTN gene was excluded. This report expands the geographical region of incidence and encourages additional studies to clarify the genetic heterogeneity of the condition. Copyright © 2010 Elsevier B.V. All rights reserved.

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

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

Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp

2013-11-29

Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143more » flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/K{sub app} reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.« less

A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers

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

Glodzik, Dominik; Morganella, Sandro; Davies, Helen

Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. Furthermore, the transcriptomicmore » consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.« less

A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers

DOE PAGES

Glodzik, Dominik; Morganella, Sandro; Davies, Helen; ...

2017-01-23

Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. Furthermore, the transcriptomicmore » consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.« less

Mutations in riboflavin transporter present with severe sensory loss and deafness in childhood.

PubMed

Srour, Myriam; Putorti, Maria Lisa; Schwartzentruber, Jeremy; Bolduc, Véronique; Shevell, Michael Israel; Poulin, Chantal; O'ferrall, Erin; Buhas, Daniela; Majewski, Jacek; Brais, Bernard

2014-11-01

We have identified a large consanguineous Lebanese family with 5 individuals with severe childhood-onset recessive sensory loss associated with deafness and variable optic atrophy. Autozygosity mapping was performed in all affected individuals, followed by whole-exome sequencing (WES) in 2 individuals. WES identified a homozygous missense mutation (c.916G>A, p.G306R) in the cerebral riboflavin transporter SLC52A2, recently shown to cause Brown-Vialetto-Van-Laere syndrome (BVVLS), which is considered primarily a motor neuronopathy. Our patients have a phenotype distinct from BVVLS, characterized by severe progressive sensory loss mainly affecting vibration and proprioception that evolves to include sensorineural hearing loss in childhood, variable degrees of optic atrophy, and marked upper extremity weakness and atrophy. Treatment of 3 patients with 400 mg/day riboflavin over 3 months produced definite clinical improvement. Mutations in SLC52A2 result in a recognizable phenotype distinct from BVVLS. Early recognition of this disorder is critical, given its potential treatability. © 2014 Wiley Periodicals, Inc.

Glyphosate resistance: state of knowledge

PubMed Central

Sammons, Robert Douglas; Gaines, Todd A

2014-01-01

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

The role of weak selection and high mutation rates in nearly neutral evolution.

PubMed

Lawson, Daniel John; Jensen, Henrik Jeldtoft

2009-04-21

Neutral dynamics occur in evolution if all types are 'effectively equal' in their reproductive success, where the definition of 'effectively equal' depends on the population size and the details of mutations. Empirically observed neutral genetic evolution in extremely large clonal populations can only be explained under current models if selection is completely absent. Such models typically consider the case where population dynamics occurs on a different timescale to evolution. However, this assumption is invalid when mutations are not rare in a whole population. We show that this has important consequences for the occurrence of neutral evolution in clonal populations. In highly connected type spaces, neutral dynamics can occur for all population sizes despite significant selective differences, via the forming of effectively neutral networks connecting rare neutral types. Biological implications include an explanation for the high diversity of rare types that survive in large clonal populations, and a theoretical justification for the use of neutral null models.

Active site remodeling switches HIV specificity of antiretroviral TRIMCyp

PubMed Central

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

2011-01-01

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

Muscle cell and motor protein function in patients with a IIa myosin missense mutation (Glu-706 to Lys).

PubMed

Li, M; Lionikas, A; Yu, F; Tajsharghi, H; Oldfors, A; Larsson, L

2006-11-01

The pathogenic events leading to the progressive muscle weakness in patients with a E706K mutation in the head of the myosin heavy chain (MyHC) IIa were analyzed at the muscle cell and motor protein levels. Contractile properties were measured in single muscle fiber segments using the skinned fiber preparation and a single muscle fiber in vitro motility assay. A dramatic impairment in the function of the IIa MyHC isoform was observed at the motor protein level. At the single muscle fiber level, on the other hand, a general decrease was observed in the number of preparations where the specific criteria for acceptance were fulfilled irrespective of MyHC isoform expression. Our results provide evidence that the pathogenesis of the MyHC IIa E706K myopathy involves defective function of the mutated myosin as well as alterations in the structural integrity of all muscle cells irrespective of MyHC isoform expression.

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 6 2011-07-01 2011-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 6 2010-07-01 2010-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 6 2012-07-01 2012-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 6 2014-07-01 2014-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 6 2013-07-01 2013-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

Validation of next generation sequencing technologies in comparison to current diagnostic gold standards for BRAF, EGFR and KRAS mutational analysis.

PubMed

McCourt, Clare M; McArt, Darragh G; Mills, Ken; Catherwood, Mark A; Maxwell, Perry; Waugh, David J; Hamilton, Peter; O'Sullivan, Joe M; Salto-Tellez, Manuel

2013-01-01

Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.

Inference of the Distribution of Selection Coefficients for New Nonsynonymous Mutations Using Large Samples

PubMed Central

Kim, Bernard Y.; Huber, Christian D.; Lohmueller, Kirk E.

2017-01-01

The distribution of fitness effects (DFE) has considerable importance in population genetics. To date, estimates of the DFE come from studies using a small number of individuals. Thus, estimates of the proportion of moderately to strongly deleterious new mutations may be unreliable because such variants are unlikely to be segregating in the data. Additionally, the true functional form of the DFE is unknown, and estimates of the DFE differ significantly between studies. Here we present a flexible and computationally tractable method, called Fit∂a∂i, to estimate the DFE of new mutations using the site frequency spectrum from a large number of individuals. We apply our approach to the frequency spectrum of 1300 Europeans from the Exome Sequencing Project ESP6400 data set, 1298 Danes from the LuCamp data set, and 432 Europeans from the 1000 Genomes Project to estimate the DFE of deleterious nonsynonymous mutations. We infer significantly fewer (0.38–0.84 fold) strongly deleterious mutations with selection coefficient |s| > 0.01 and more (1.24–1.43 fold) weakly deleterious mutations with selection coefficient |s| < 0.001 compared to previous estimates. Furthermore, a DFE that is a mixture distribution of a point mass at neutrality plus a gamma distribution fits better than a gamma distribution in two of the three data sets. Our results suggest that nearly neutral forces play a larger role in human evolution than previously thought. PMID:28249985

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes

PubMed Central

Piluso, G; Politano, L; Aurino, S; Fanin, M; Ricci, E; Ventriglia, V; Belsito, A; Totaro, A; Saccone, V; Topaloglu, H; Nascimbeni, A; Fulizio, L; Broccolini, A; Canki-Klain, N; Comi, L; Nigro, G; Angelini, C; Nigro, V

2005-01-01

Background: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. Objective: To obtain unbiased information on the consequences of CAPN3 mutations. Patients: 530 subjects with different grades of symptoms and 300 controls. Methods: High throughput denaturing HPLC analysis of DNA pools. Results: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. Conclusions: A non-invasive and cost–effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103. PMID:16141003

Xeroderma Pigmentosum-Trichothiodystrophy overlap patient with novel XPD/ERCC2 mutation

PubMed Central

Kralund, Henrik H.; Ousager, Lilian; Jaspers, Nicolaas G.; Raams, Anja; Pedersen, Erling B.; Gade, Else; Bygum, Anette

2013-01-01

Xeroderma Pigmentosum (XP), Trichothiodystrophy (TTD) and Cockayne Syndrome (CS) are rare, recessive disorders caused by mutational defects in the Nucleotide Excision Repair (NER) pathway and/or disruption of basic cellular DNA transcription. To date, a multitude of mutations in the XPD/ERCC2 gene have been described, many of which give rise to NER- and DNA transcription related diseases, which share certain diagnostic features and few overlap patients have been described. Despite increasing understanding of the roles of XPD/ERCC2 in mammalian cells, there is still weak predictability of somatic outcome from many of these mutations. We demonstrate a patient, believed to represent an overlap between XP and TTD/CS. In addition to other organ dysfunctions, the young man presented with Photosensitivity, Ichthyosis, Brittle hair, Impaired physical and mental development, Decreased fertility and Short stature (PIBIDS) suggestive of TTD, but lacking the almost patognomonic “tiger tail” banding of the hair under polarized light. Additionally, he developed basal cell carcinoma aged 28, as well as adult onset kidney failure, features normally not associated with TTD but rather XP/CS. His freckled appearance also suggested XP, but fibroblast cultures only demonstrated x2 UV-sensitivity with expected NER and TFIIH-activity decrease. Genetic sequencing of the XPD/ERCC2 gene established the patient as heterozygote compound with a novel, N-terminal Y18H mutation and a known C-terminal (TTD) mutation, A725P. The possible interplay between gene products and the patient phenotype is discussed. PMID:25002996

The expanding phenotype of mitochondrial myopathy.

PubMed

DiMauro, Salvatore; Gurgel-Giannetti, Juliana

2005-10-01

Our understanding of mitochondrial diseases (defined restrictively as defects in the mitochondrial respiratory chain) continues to progress apace. In this review we provide an update of information regarding disorders that predominantly or exclusively affect skeletal muscle. Most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency, and mutations in genes that control mitochondrial DNA (mtDNA) abundance and structure such as POLG and TK2. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with altered lipid composition of the inner mitochondrial membrane, but a putative secondary impairment of the respiratory chain remains to be documented. Concerning the 'other genome', the role played by mutations in protein encoding genes of mtDNA in causing isolated myopathies has been confirmed. It has also been confirmed that mutations in tRNA genes of mtDNA can cause predominantly myopathic syndromes and - contrary to conventional wisdom - these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, myalgia, cramps, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

Deleterious mutations and selection for sex in finite diploid populations.

PubMed

Roze, Denis; Michod, Richard E

2010-04-01

In diploid populations, indirect benefits of sex may stem from segregation and recombination. Although it has been recognized that finite population size is an important component of selection for recombination, its effects on selection for segregation have been somewhat less studied. In this article, we develop analytical two- and three-locus models to study the effect of recurrent deleterious mutations on a modifier gene increasing sex, in a finite diploid population. The model also incorporates effects of mitotic recombination, causing loss of heterozygosity (LOH). Predictions are tested using multilocus simulations representing deleterious mutations occurring at a large number of loci. The model and simulations show that excess of heterozygosity generated by finite population size is an important component of selection for sex, favoring segregation when deleterious alleles are nearly additive to dominant. Furthermore, sex tends to break correlations in homozygosity among selected loci, which disfavors sex when deleterious alleles are either recessive or dominant. As a result, we find that it is difficult to maintain costly sex when deleterious alleles are recessive. LOH tends to favor sex when deleterious mutations are recessive, but the effect is relatively weak for rates of LOH corresponding to current estimates (of the order 10(-4)-10(-5)).

Genetic Diversity in the Interference Selection Limit

PubMed Central

Good, Benjamin H.; Walczak, Aleksandra M.; Neher, Richard A.; Desai, Michael M.

2014-01-01

Pervasive natural selection can strongly influence observed patterns of genetic variation, but these effects remain poorly understood when multiple selected variants segregate in nearby regions of the genome. Classical population genetics fails to account for interference between linked mutations, which grows increasingly severe as the density of selected polymorphisms increases. Here, we describe a simple limit that emerges when interference is common, in which the fitness effects of individual mutations play a relatively minor role. Instead, similar to models of quantitative genetics, molecular evolution is determined by the variance in fitness within the population, defined over an effectively asexual segment of the genome (a “linkage block”). We exploit this insensitivity in a new “coarse-grained” coalescent framework, which approximates the effects of many weakly selected mutations with a smaller number of strongly selected mutations that create the same variance in fitness. This approximation generates accurate and efficient predictions for silent site variability when interference is common. However, these results suggest that there is reduced power to resolve individual selection pressures when interference is sufficiently widespread, since a broad range of parameters possess nearly identical patterns of silent site variability. PMID:24675740

Imprints from genetic drift and mutation imply relative divergence times across marine transition zones in a pan-European small pelagic fish (Sprattus sprattus).

PubMed

Limborg, M T; Hanel, R; Debes, P V; Ring, A K; André, C; Tsigenopoulos, C S; Bekkevold, D

2012-08-01

Geographic distributions of most temperate marine fishes are affected by postglacial recolonisation events, which have left complex genetic imprints on populations of marine species. This study investigated population structure and demographic history of European sprat (Sprattus sprattus L.) by combining inference from both mtDNA and microsatellite genetic markers throughout the species' distribution. We compared effects from genetic drift and mutation for both genetic markers in shaping genetic differentiation across four transition zones. Microsatellite markers revealed significant isolation by distance and a complex population structure across the species' distribution (overall θ(ST)=0.038, P<0.01). Across transition zones markers indicated larger effects of genetic drift over mutations in the northern distribution of sprat contrasting a stronger relative impact of mutation in the species' southern distribution in the Mediterranean region. These results were interpreted to reflect more recent divergence times between northern populations in accordance with previous findings. This study demonstrates the usefulness of comparing inference from different markers and estimators of divergence for phylogeographic and population genetic studies in species with weak genetic structure, as is the case in many marine species.

Continuous and discontinuous phase transitions in the evolution of a polygenic trait under stabilizing selective pressure

NASA Astrophysics Data System (ADS)

Fierro, Annalisa; Cocozza, Sergio; Monticelli, Antonella; Scala, Giovanni; Miele, Gennaro

2017-06-01

The presence of phenomena analogous to phase transition in Statistical Mechanics has been suggested in the evolution of a polygenic trait under stabilizing selection, mutation and genetic drift. By using numerical simulations of a model system, we analyze the evolution of a population of N diploid hermaphrodites in random mating regime. The population evolves under the effect of drift, selective pressure in form of viability on an additive polygenic trait, and mutation. The analysis allows to determine a phase diagram in the plane of mutation rate and strength of selection. The involved pattern of phase transitions is characterized by a line of critical points for weak selective pressure (smaller than a threshold), whereas discontinuous phase transitions, characterized by metastable hysteresis, are observed for strong selective pressure. A finite-size scaling analysis suggests the analogy between our system and the mean-field Ising model for selective pressure approaching the threshold from weaker values. In this framework, the mutation rate, which allows the system to explore the accessible microscopic states, is the parameter controlling the transition from large heterozygosity ( disordered phase) to small heterozygosity ( ordered one).

Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand.

PubMed

Yuan, Ji; Cheung, Paul K M; Zhang, Huifang M; Chau, David; Yang, Decheng

2005-02-01

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

PubMed Central

Amyot, Whitney M.; deJesus, Dennise

2013-01-01

Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system. PMID:23798536

Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4 or Rab11 pathway.

PubMed

Wang, Feifei; Chen, Xiaoqing; Zhang, Xiaoqing; Ma, Lan

2008-08-01

Agonist-induced phosphorylation, internalization, and intracellular trafficking of G protein-coupled receptors are critical in regulating both cellular responsiveness and signal transduction. The current study investigated the role of receptor phosphorylation state in regulation of agonist-induced internalization and intracellular trafficking of mu-opioid receptor (MOR). Our results showed that after agonist stimulation, the recycle of a mutant MOR that lacks the C-terminal residues after Asn(362) (MOR362T) was greatly decreased, whereas a C-terminal phosphorylation sites-mutated MOR (MOR3A), which is deficient in agonist-induced phosphorylation recycled back to the membrane at a level comparable to that of the wild-type receptor, however, interestingly at a slower rate. Inhibition of functions of either Rab4 or Rab11 by dominant-negative mutants and small interfering RNA both significantly impaired the recycling of the wild-type MOR, whereas the recycling of the phosphorylation-deficient mutant was only inhibited by the dominant-negative mutant and small interfering RNA of Rab11, suggesting that the recycling of nonphosphorylated MOR is exclusively via Rab11-mediated pathway. Furthermore, phosphorylated MOR was observed accumulated in Rab5- and Rab4-, but not Rab11-positive vesicles. Our data indicate that both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation, and the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively, which may ultimately lead to differential cellular responsiveness or downstream signaling.

Colored petri net modeling of small interfering RNA-mediated messenger RNA degradation.

PubMed

Nickaeen, Niloofar; Moein, Shiva; Heidary, Zarifeh; Ghaisari, Jafar

2016-01-01

Mathematical modeling of biological systems is an attractive way for studying complex biological systems and their behaviors. Petri Nets, due to their ability to model systems with various levels of qualitative information, have been wildly used in modeling biological systems in which enough qualitative data may not be at disposal. These nets have been used to answer questions regarding the dynamics of different cell behaviors including the translation process. In one stage of the translation process, the RNA sequence may be degraded. In the process of degradation of RNA sequence, small-noncoding RNA molecules known as small interfering RNA (siRNA) match the target RNA sequence. As a result of this matching, the target RNA sequence is destroyed. In this context, the process of matching and destruction is modeled using Colored Petri Nets (CPNs). The model is constructed using CPNs which allow tokens to have a value or type on them. Thus, CPN is a suitable tool to model string structures in which each element of the string has a different type. Using CPNs, long RNA, and siRNA strings are modeled with a finite set of colors. The model is simulated via CPN Tools. A CPN model of the matching between RNA and siRNA strings is constructed in CPN Tools environment. In previous studies, a network of stoichiometric equations was modeled. However, in this particular study, we modeled the mechanism behind the silencing process. Modeling this kind of mechanisms provides us with a tool to examine the effects of different factors such as mutation or drugs on the process.

Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

PubMed

Jeudy, Jeremy; Salvador, Arnaud; Simon, Romain; Jaffuel, Aurore; Fonbonne, Catherine; Léonard, Jean-François; Gautier, Jean-Charles; Pasquier, Olivier; Lemoine, Jerome

2014-02-01

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 μg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

200-GHz and 50-GHz AWG channelized linewidth dependent transmission of weak-resonant-cavity FPLD injection-locked by spectrally sliced ASE.

PubMed

Lin, Gong-Ru; Cheng, Tzu-Kang; Chi, Yu-Chieh; Lin, Gong-Cheng; Wang, Hai-Lin; Lin, Yi-Hong

2009-09-28

In a weak-resonant-cavity Fabry-Perot laser diode (WRC-FPLD) based DWDM-PON system with an array-waveguide-grating (AWG) channelized amplified spontaneous emission (ASE) source located at remote node, we study the effect of AWG filter bandwidth on the transmission performances of the 1.25-Gbit/s directly modulated WRC-FPLD transmitter under the AWG channelized ASE injection-locking. With AWG filters of two different channel spacings at 50 and 200 GHz, several characteristic parameters such as interfered reflection, relatively intensity noise, crosstalk reduction, side-mode-suppressing ratio and power penalty of BER effect of the WRC-FPLD transmitted data are compared. The 200-GHz AWG filtered ASE injection minimizes the noises of WRC-FPLD based ONU transmitter, improving the power penalty of upstream data by -1.6 dB at BER of 10(-12). In contrast, the 50-GHz AWG channelized ASE injection fails to promote better BER but increases the power penalty by + 1.5 dB under back-to-back transmission. A theoretical modeling elucidates that the BER degradation up to 4 orders of magnitude between two injection cases is mainly attributed to the reduction on ASE injection linewidth, since which concurrently degrades the signal-to-noise and extinction ratios of the transmitted data stream.

32 CFR 234.6 - Interfering with agency functions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 2 2010-07-01 2010-07-01 false Interfering with agency functions. 234.6 Section 234.6 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS CONDUCT ON THE PENTAGON RESERVATION § 234.6 Interfering with agency functions. The following are...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 4 2010-10-01 2010-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 4 2011-10-01 2011-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 4 2014-10-01 2014-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 4 2013-10-01 2013-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 4 2012-10-01 2012-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

Incorporating information about pre-implantation genetic diagnosis into discussions about testing and risk-management for BRCA1/2 mutations: A qualitative study of patient preferences

PubMed Central

Hurley, Karen; Rubin, Lisa; Werner-Lin, Allison; Sagi, Michal; Kemel, Yelena; Stern, Rikki; Phillips, Aliza; Cholst, Ina; Kauff, Noah; Offit, Kenneth

2016-01-01

Background Studies show that BRCA1/2 mutation carriers are interested in learning about reproductive options such as pre-implantation genetic diagnosis (PGD) to prevent passing their risk onto their children. However, attitudes vary widely, and the procedure raises complex ethical and psychosocial issues. This complexity, plus the highly technical nature of PGD, makes it difficult to integrate PGD information into genetic counseling sessions that already cover probabilistic, emotionally-charged risk information. Method Thirty-three reproductive age BRCA1/2 mutation carriers who had previously undergone genetic counseling viewed a tutorial about PGD and were interviewed about attitudes towards PGD, and preferences about how to include PGD information in genetic counseling. Results Most participants preferred to be briefly informed of availability of PGD information, and to receive written materials about PGD, but with the option of deferring detailed discussion if they already feel overloaded or perceive that PGD is not immediately relevant to their risk management and/or childbearing plans. For some, the stress of testing temporarily interfered with information processing, producing states of cognitive avoidance (“in a fog,” “tuning out”). Some preferred to discuss PGD with a physician with whom they had an ongoing relationship (e.g., OB/GYN, primary care provider, oncologist). Conclusions Providers offering cancer genetic testing can consider indicating availability of PGD information, while attending to patients’ level of interest and ability to absorb information. Research is needed to link patient responses to information overload to psychosocial outcomes (e.g., distress, decision quality). Continuing medical education is needed to support providers in facilitating informed decisions about PGD. PMID:22736296

An RNA-splicing mutation (G{sup +51VS20}) in the Type II collagen gene (COL2A1) in a family with spondyloepiphyseal dysplasia congenita

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

Tiller, G.E.; Polumbo, P.A.; Weis, M.A.

1995-02-01

Defects in type II collagen have been demonstrated in a phenotypic continuum of chondrodysplasias that includes achondrogenesis II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, and Stickler syndrome. We have determined that cartilage from a terminated fetus with an inherited form of SEDC contained both normal {alpha}1(II) collagen chains and chains that lacked amino acids 256-273 of the triple-helical domain. PCR amplification of this region of COL2A1, from genomic DNA, yielded products of normal size, while amplification of cDNA yielded a normal sized species and a shorter fragment missing exon 20. Sequence analysis of genomic DNA from the fetus revealedmore » a G{yields}T transversion at position +5 of intron 20; the affected father was also heterozygous for the mutation. Allele-specific PCR and heteroduplex analysis of a VNTR in COL2A1 independently confirmed the unaffected status of a fetus in a subsequent pregnancy. Thermodynamic calculations suggest that the mutation prevents normal splicing of exon 20 by interfering with binding of U{sub 1} small-nuclear RNA to pre-mRNA, thus leading to skipping of exon 20 in transcripts from the mutant allele. Electron micrographs of diseased cartilage showed intracellular inclusion bodies, which were stained by an antibody to {alpha}1(II) procollagen. Our findings support the hypothesis that {alpha}-chain length alterations that preserve the Gly-X-Y repeat motif of the triple helix result in partial intracellular retention of {alpha}1(II) procollagen and produce mild to moderate chondrodysplasia phenotypes. 50 refs., 6 figs., 1 tab.« less

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

Adult cases of mitochondrial DNA depletion due to TK2 defect: an expanding spectrum.

PubMed

Béhin, A; Jardel, C; Claeys, K G; Fagart, J; Louha, M; Romero, N B; Laforêt, P; Eymard, B; Lombès, A

2012-02-28

In this study we aim to demonstrate the occurrence of adult forms of TK2 mutations causing progressive mitochondrial myopathy with significant muscle mitochondrial DNA (mtDNA) depletion. Patients' investigations included serum creatine kinase, blood lactate, electromyographic, echocardiographic, and functional respiratory analyses as well as TK2 gene sequencing and TK2 activity measurement. Mitochondrial activities and mtDNA were analyzed in the patients' muscle biopsy. The 3 adult patients with TK2 mutations presented with slowly progressive myopathy compatible with a fairly normal life during decades. Apart from its much slower progression, these patients' phenotype closely resembled that of pediatric cases including early onset, absence of CNS symptoms, generalized muscle weakness predominating on axial and proximal muscles but affecting facial, ocular, and respiratory muscles, typical mitochondrial myopathy with a mosaic pattern of COX-negative and ragged-red fibers, combined mtDNA-dependent respiratory complexes deficiency and mtDNA depletion. In accordance with the disease's relatively slow progression, the residual mtDNA content was higher than that observed in pediatric cases. That difference was not explained by the type of the TK2 mutations or by the residual TK2 activity. TK2 mutations can cause mitochondrial myopathy with a slow progression. Comparison of patients with similar mutations but different disease progression might address potential mechanisms of mtDNA maintenance modulation.

Novel SIL1 nonstop mutation in a Chinese consanguineous family with Marinesco-Sjögren syndrome and Dandy-Walker syndrome.

PubMed

Gai, Nan; Jiang, Chen; Zou, Yong-Yi; Zheng, Yu; Liang, De-Sheng; Wu, Ling-Qian

2016-07-01

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive disorder, which is characterized by congenital cataracts, cerebellar ataxia, progressive muscle weakness, and delayed psychomotor development. SIL1, which is located at 5q31.2, is the only gene known to cause MSS. Dandy-Walker syndrome (DWS) is defined by hypoplasia, upward rotation of the cerebellar vermis, and cystic dilation of the fourth ventricle; however, its genetic pathogeny remains unclear. Here, we report a Chinese consanguineous family with MSS and DWS. Whole exome sequencing identified a novel nonstop mutation in SIL1. Sanger sequencing revealed that the mutation was segregated in this family according to a recessive mode of inheritance. We found that the mutation changed a stop codon (TGA) to an arginine codon (CGA), and no in-frame termination codon in the 3' untranslated region (UTR) of SIL1 could be found. The mRNA levels of SIL1 were decreased by 56.6% and 37.5% in immortalized lymphoblasts of the patients respectively; the protein levels of SIL1 were substantially decreased. This case study is the first report on Chinese MSS patients, MSS complicated by DWS, and a nonstop mutation in SIL1. Our findings imply the pathogenetic association between DWS and MSS. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Novel VCP mutations in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia.

PubMed

Watts, G D J; Thomasova, D; Ramdeen, S K; Fulchiero, E C; Mehta, S G; Drachman, D A; Weihl, C C; Jamrozik, Z; Kwiecinski, H; Kaminska, A; Kimonis, V E

2007-11-01

Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD, OMIM 167320) has recently been attributed to eight missense mutations in valosin-containing protein (VCP). We report novel VCP mutations N387H and L198W in six individuals from two families who presented with proximal muscle weakness at a mean age of diagnosis of 40 years, most losing the ability to walk within a few years of onset. Electromyographic studies in four individuals were suggestive of 'myopathic' changes, and neuropathic pattern was identified in one individual in family 1. Muscle biopsy in four individuals showed myopathic changes characterized by variable fiber size, two individuals showing rimmed vacuoles and IBM-type cytoplasmic inclusions in muscle fibers, and electron microscopy in one individual revealing abundant intranuclear inclusions. Frontotemporal dementia associated with characteristic behavioral changes including short-term memory loss, language difficulty, and antisocial behavior was observed in three individuals at a mean age of 47 years. Detailed brain pathology in one individual showed cortical degenerative changes, most severe in the temporal lobe and hippocampus. Abundant ubiquitin-positive tau-, alpha-synuclein-, polyglutamine repeat-negative neuronal intranuclear inclusions and only rare intracytoplasmic VCP positive inclusions were seen. These new mutations may cause structural changes in VCP and provide some insight into the functional effects of pathogenic mutations.

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2014 CFR

2014-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2013 CFR

2013-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2012 CFR

2012-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2011 CFR

2011-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2010 CFR

2010-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

Fine mapping for Weaver Syndrome in the Brown Swiss breed with the identification of possible casual mutations across NRCAM, PNPLA8, and CTTNBP2 and Developement of a diagnostic SNP haplotype

USDA-ARS?s Scientific Manuscript database

Bovine Progressive Degenerative Myeloencephalopathy (Weaver Syndrome) is a recessive neurological disease that has been observed in the Brown Swiss cattle breed since the 1970’s in North America and Europe. Bilateral hind leg weakness and ataxia appear in afflicted animals at 6 to 18 months of age,...

CORRELATION OF CARCINOGENIC POTENCY WITH MOUSE SKIN 32P-POSTLABELING AND LAC Z-MUTATION DATE FOR DMBA AN ITS K-REGION SULPHUR ISOSTERE: COMPARISON WITH ACTIVITIES OBSERVED IN STANDARD GENOTOXICITY ASSAYS

EPA Science Inventory

6,11-Dimethylbenzo(b]naphtho[2,3-d]thiophene (S-DMBA) is one of several carcinogenic analogs of the reference mouse skin carcinogen 7,12-dimethylbenz[alanthracene (OMBA)Demonstration of the weak carcinogenicity of S-DMBA by Tilak in 1946 established at that early stage the inadeq...

Analyses of Evolutionary Characteristics of the Hemagglutinin-Esterase Gene of Influenza C Virus during a Period of 68 Years Reveals Evolutionary Patterns Different from Influenza A and B Viruses.

PubMed

Furuse, Yuki; Matsuzaki, Yoko; Nishimura, Hidekazu; Oshitani, Hitoshi

2016-11-26

Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1) multiple lineages have been circulating globally; (2) there have been weak and infrequent selective bottlenecks; (3) the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4) there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics.

Analyses of Evolutionary Characteristics of the Hemagglutinin-Esterase Gene of Influenza C Virus during a Period of 68 Years Reveals Evolutionary Patterns Different from Influenza A and B Viruses

PubMed Central

Furuse, Yuki; Matsuzaki, Yoko; Nishimura, Hidekazu; Oshitani, Hitoshi

2016-01-01

Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1) multiple lineages have been circulating globally; (2) there have been weak and infrequent selective bottlenecks; (3) the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4) there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics. PMID:27898037

Dropped head congenital muscular dystrophy caused by de novo mutations in LMNA.

PubMed

Karaoglu, Pakize; Quizon, Nicolas; Pergande, Matthias; Wang, Haicui; Polat, Ayşe Ipek; Ersen, Ayca; Özer, Erdener; Willkomm, Lena; Hiz Kurul, Semra; Heredia, Raúl; Yis, Uluç; Selcen, Duygu; Çirak, Sebahattin

2017-04-01

Dropped head syndrome is an easily recognizable clinical presentation of Lamin A/C-related congenital muscular dystrophy. Patients usually present in the first year of life with profound neck muscle weakness, dropped head, and elevated serum creatine kinase. Two patients exhibited head drop during infancy although they were able to sit independently. Later they developed progressive axial and limb-girdle weakness. Creatine kinase levels were elevated and muscle biopsies of both patients showed severe dystrophic changes. The distinctive clinical hallmark of the dropped head led us to the diagnosis of Lamin A/C-related congenital muscular dystrophy, with a pathogenic de novo mutation p.Glu31del in the head domain of the Lamin A/C gene in both patients. Remarkably, one patient also had a central involvement with white matter changes on brain magnetic resonance imaging. Lamin A/C-related dropped-head syndrome is a rapidly progressive congenital muscular dystrophy and may lead to loss of ambulation, respiratory insufficiency, and cardiac complications. Thus, the genetic diagnosis of dropped-head syndrome as L-CMD and the implicated clinical care protocols are of vital importance for these patients. This disease may be underdiagnosed, as only a few genetically confirmed cases have been reported. Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Allele Age Under Non-Classical Assumptions is Clarified by an Exact Computational Markov Chain Approach.

PubMed

De Sanctis, Bianca; Krukov, Ivan; de Koning, A P Jason

2017-09-19

Determination of the age of an allele based on its population frequency is a well-studied problem in population genetics, for which a variety of approximations have been proposed. We present a new result that, surprisingly, allows the expectation and variance of allele age to be computed exactly (within machine precision) for any finite absorbing Markov chain model in a matter of seconds. This approach makes none of the classical assumptions (e.g., weak selection, reversibility, infinite sites), exploits modern sparse linear algebra techniques, integrates over all sample paths, and is rapidly computable for Wright-Fisher populations up to N e  = 100,000. With this approach, we study the joint effect of recurrent mutation, dominance, and selection, and demonstrate new examples of "selective strolls" where the classical symmetry of allele age with respect to selection is violated by weakly selected alleles that are older than neutral alleles at the same frequency. We also show evidence for a strong age imbalance, where rare deleterious alleles are expected to be substantially older than advantageous alleles observed at the same frequency when population-scaled mutation rates are large. These results highlight the under-appreciated utility of computational methods for the direct analysis of Markov chain models in population genetics.

Thermodynamic Basis of Selectivity in the Interactions of Tissue Inhibitors of Metalloproteinases N-domains with Matrix Metalloproteinases-1, -3, and -14*

PubMed Central

Zou, Haiyin; Wu, Ying

2016-01-01

The four tissue inhibitors of metalloproteinases (TIMPs) are potent inhibitors of the many matrixins (MMPs), except that TIMP1 weakly inhibits some MMPs, including MMP14. The broad-spectrum inhibition of MMPs by TIMPs and their N-domains (NTIMPs) is consistent with the previous isothermal titration calorimetric finding that their interactions are entropy-driven but differ in contributions from solvent and conformational entropy (ΔSsolv, ΔSconf), estimated using heat capacity changes (ΔCp). Selective engineered NTIMPs have potential applications for treating MMP-related diseases, including cancer and cardiomyopathy. Here we report isothermal titration calorimetric studies of the effects of selectivity-modifying mutations in NTIMP1 and NTIMP2 on the thermodynamics of their interactions with MMP1, MMP3, and MMP14. The weak inhibition of MMP14 by NTIMP1 reflects a large conformational entropy penalty for binding. The T98L mutation, peripheral to the NTIMP1 reactive site, enhances binding by increasing ΔSsolv but also reduces ΔSconf. However, the same mutation increases NTIMP1 binding to MMP3 in an interaction that has an unusual positive ΔCp. This indicates a decrease in solvent entropy compensated by increased conformational entropy, possibly reflecting interactions involving alternative conformers. The NTIMP2 mutant, S2D/S4A is a selective MMP1 inhibitor through electrostatic effects of a unique MMP-1 arginine. Asp-2 increases reactive site polarity, reducing ΔCp, but increases conformational entropy to maintain strong binding to MMP1. There is a strong negative correlation between ΔSsolv and ΔSconf for all characterized interactions, but the data for each MMP have characteristic ranges, reflecting intrinsic differences in the structures and dynamics of their free and inhibitor-bound forms. PMID:27033700

Null steering of adaptive beamforming using linear constraint minimum variance assisted by particle swarm optimization, dynamic mutated artificial immune system, and gravitational search algorithm.

PubMed

Darzi, Soodabeh; Kiong, Tiong Sieh; Islam, Mohammad Tariqul; Ismail, Mahamod; Kibria, Salehin; Salem, Balasem

2014-01-01

Linear constraint minimum variance (LCMV) is one of the adaptive beamforming techniques that is commonly applied to cancel interfering signals and steer or produce a strong beam to the desired signal through its computed weight vectors. However, weights computed by LCMV usually are not able to form the radiation beam towards the target user precisely and not good enough to reduce the interference by placing null at the interference sources. It is difficult to improve and optimize the LCMV beamforming technique through conventional empirical approach. To provide a solution to this problem, artificial intelligence (AI) technique is explored in order to enhance the LCMV beamforming ability. In this paper, particle swarm optimization (PSO), dynamic mutated artificial immune system (DM-AIS), and gravitational search algorithm (GSA) are incorporated into the existing LCMV technique in order to improve the weights of LCMV. The simulation result demonstrates that received signal to interference and noise ratio (SINR) of target user can be significantly improved by the integration of PSO, DM-AIS, and GSA in LCMV through the suppression of interference in undesired direction. Furthermore, the proposed GSA can be applied as a more effective technique in LCMV beamforming optimization as compared to the PSO technique. The algorithms were implemented using Matlab program.

Null Steering of Adaptive Beamforming Using Linear Constraint Minimum Variance Assisted by Particle Swarm Optimization, Dynamic Mutated Artificial Immune System, and Gravitational Search Algorithm

PubMed Central

Sieh Kiong, Tiong; Tariqul Islam, Mohammad; Ismail, Mahamod; Salem, Balasem

2014-01-01

Linear constraint minimum variance (LCMV) is one of the adaptive beamforming techniques that is commonly applied to cancel interfering signals and steer or produce a strong beam to the desired signal through its computed weight vectors. However, weights computed by LCMV usually are not able to form the radiation beam towards the target user precisely and not good enough to reduce the interference by placing null at the interference sources. It is difficult to improve and optimize the LCMV beamforming technique through conventional empirical approach. To provide a solution to this problem, artificial intelligence (AI) technique is explored in order to enhance the LCMV beamforming ability. In this paper, particle swarm optimization (PSO), dynamic mutated artificial immune system (DM-AIS), and gravitational search algorithm (GSA) are incorporated into the existing LCMV technique in order to improve the weights of LCMV. The simulation result demonstrates that received signal to interference and noise ratio (SINR) of target user can be significantly improved by the integration of PSO, DM-AIS, and GSA in LCMV through the suppression of interference in undesired direction. Furthermore, the proposed GSA can be applied as a more effective technique in LCMV beamforming optimization as compared to the PSO technique. The algorithms were implemented using Matlab program. PMID:25147859

An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein*

PubMed Central

Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J.; Backofen, Rolf; Marchfelder, Anita

2015-01-01

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. PMID:25512373

Identification of Germline Genetic Mutations in Pancreatic Cancer Patients

PubMed Central

Salo-Mullen, Erin E.; O’Reilly, Eileen; Kelsen, David; Ashraf, Asad M.; Lowery, Maeve; Yu, Kenneth; Reidy, Diane; Epstein, Andrew S.; Lincoln, Anne; Saldia, Amethyst; Jacobs, Lauren M.; Rau-Murthy, Rohini; Zhang, Liying; Kurtz, Robert; Saltz, Leonard; Offit, Kenneth; Robson, Mark; Stadler, Zsofia K.

2016-01-01

Background Pancreatic adenocarcinoma (PAC) is part of several cancer predisposition syndromes; however, indications for genetic counseling/testing are not well-defined. We sought to determine mutation prevalence and characteristics that predict for inherited predisposition to PAC. Methods We identified 175 consecutive PAC patients who underwent clinical genetics assessment at Memorial Sloan Kettering between 2011–2014. Clinical data, family history, and germline results were evaluated. Results Among 159 PAC patients who pursued genetic testing, 24 pathogenic mutations were identified (15.1%; 95%CI, 9.5%–20.7%), including BRCA2(n=13), BRCA1(n=4), p16(n=2), PALB2(n=1), and Lynch syndrome(n=4). BRCA1/BRCA2 prevalence was 13.7% in Ashkenazi Jewish(AJ) (n=95) and 7.1% in non-AJ(n=56) patients. In AJ patients with strong, weak, or absent family history of BRCA-associated cancers, mutation prevalence was 16.7%, 15.8%, and 7.4%, respectively. Mean age at diagnosis in all mutation carriers was 58.5y(range 45–75y) compared to 64y(range 27–87y) in non-mutation carriers(P=0.02). Although BRCA2 was the most common mutation identified, no patients with early-onset PAC(≤50y) harbored a BRCA2 mutation and the mean age at diagnosis in BRCA2 carriers was equivalent to non-mutation carriers(P=0.34). Mutation prevalence in early-onset patients(n=21) was 28.6%, including BRCA1(n=2), p16(n=2), MSH2(n=1) and MLH1(n=1). Conclusion Mutations in BRCA2 account for over 50% of PAC patients with an identified susceptibility syndrome. AJ patients had high BRCA1/BRCA2 prevalence regardless of personal/family history, suggesting that ancestry alone indicates a need for genetic evaluation. With the exception of BRCA2-associated PAC, inherited predisposition to PAC is associated with earlier age at PAC diagnosis suggesting that this subset of patients may also represent a population warranting further evaluation. PMID:26440929

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

NASA Astrophysics Data System (ADS)

Vriend, Gert; Eijsink, Vincent

1993-08-01

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cercus. Several new techniques have been developed to improve the model-building procedures. Also a model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

Development of a tunable Fabry-Perot interferometer UV camera for monitoring sulfur dioxide emissions

NASA Astrophysics Data System (ADS)

Tamminen, J.; Kujanpää, J.; Ojanen, H.; Saari, H.; Näkki, I.; Tukiainen, S.; Kyrölä, E.

2017-12-01

We present a novel UV camera for sulfur dioxide emission monitoring.The camera is equipped with a piezo-actuated Fabry-Perot interferometer allowing thefilter transmission to be tuned to match the differential absorption features ofsulfur dioxide in the wavelength region 305-320 nm. The differential absorption structuresare exploited to reduce the interfering effects of weakly wavelength dependent absorbers, suchas aerosols and black carbon, present in the exhaust gas. A data processing algorithm basedon two air gaps of the filter is presented allowing collection of a sufficient signal-to-noise ratio fordetecting sulfur dioxide in the ship plumes even in the designated emission control areas, such as the Baltic Seawhere the sulfur content limit of fuel oil is 0.1 %. First field tests performed inLänsisatama harbour, Helsinki Finland, indicate that sulfur dioxide can be detectedin ship plumes. The camera is light-weight and can be mounted to a drone.

Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

PubMed Central

Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

2008-01-01

We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promising drug candidates. These molecules, in conjunction with triplex forming oligonucleotides, could potentially be used to achieve control of gene expression by interfering with transcription factors that bind to DNA. Therefore, the ability to screen for these molecules in a high-throughput fashion could dramatically improve the drug screening process. The assay reported here provides excellent discrimination between strong, intermediate, and weak duplex and triplex DNA binders in a high-throughput fashion. PMID:17614366

New laser system for highly sensitive clinical pulse oximetry

NASA Astrophysics Data System (ADS)

Hamza, Mostafa; Hamza, Mohammad

1996-04-01

This paper describes the theory and design of a new pulse oximeter in which laser diodes and other compact laser sources are used for the measurement of oxygen saturation in patients who are at risk of developing hypoxemia. The technique depends upon illuminating special sites of the skin of the patient with radiation from modulated laser sources at selected wavelengths. The specific laser wavelengths are chosen based on the absorption characteristics of oxyhemoglobin, reduced hemoglobin and other interfering sources for obtaining more accurate measurements. The laser radiation transmitted through the tissue is detected and signal processing based on differential absorption laser spectroscopy is done in such a way to overcome the primary performance limitations of the conventionally used pulse oximetry. The new laser pulse oximeter can detect weak signals and is not affected by other light sources such as surgical lamps, phototherapy units, etc. The detailed description and operating characteristics of this system are presented.

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

Alfonso Hernandez, Laura; Nelson, Tammie Renee; Gelin, Maxim F.

The interchromophoric energy-transfer pathways between weakly coupled units in a π-conjugated phenylene–ethynylene macrocycle and its half-ring analogue have been investigated using the nonadiabatic excited-state molecular dynamics approach. To track the flow of electronic transition density between macrocycle units, we formulate a transition density flux analysis adapted from the statistical minimum flow method previously developed to investigate vibrational energy flow. Following photoexcitation, transition density is primarily delocalized on two chromophore units and the system undergoes ultrafast energy transfer, creating a localized excited state on a single unit. In the macrocycle, distinct chromophore units donate transition density to a single acceptor unitmore » but do not interchange transition density among each other. We find that energy transfer in the macrocycle is slower than in the corresponding half ring because of the presence of multiple interfering energy-transfer pathways. Finally, simulation results are validated by modeling the fluorescence anisotropy decay.« less

Interference of interchromophoric energy-transfer pathways in π-conjugated macrocycles

DOE PAGES

Alfonso Hernandez, Laura; Nelson, Tammie Renee; Gelin, Maxim F.; ...

2016-11-10

The interchromophoric energy-transfer pathways between weakly coupled units in a π-conjugated phenylene–ethynylene macrocycle and its half-ring analogue have been investigated using the nonadiabatic excited-state molecular dynamics approach. To track the flow of electronic transition density between macrocycle units, we formulate a transition density flux analysis adapted from the statistical minimum flow method previously developed to investigate vibrational energy flow. Following photoexcitation, transition density is primarily delocalized on two chromophore units and the system undergoes ultrafast energy transfer, creating a localized excited state on a single unit. In the macrocycle, distinct chromophore units donate transition density to a single acceptor unitmore » but do not interchange transition density among each other. We find that energy transfer in the macrocycle is slower than in the corresponding half ring because of the presence of multiple interfering energy-transfer pathways. Finally, simulation results are validated by modeling the fluorescence anisotropy decay.« less

Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic

PubMed Central

Watts, Jonathan K.; Corey, David R.

2014-01-01

Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past twenty years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology. PMID:22069063

An Intelligent Gear Fault Diagnosis Methodology Using a Complex Wavelet Enhanced Convolutional Neural Network.

PubMed

Sun, Weifang; Yao, Bin; Zeng, Nianyin; Chen, Binqiang; He, Yuchao; Cao, Xincheng; He, Wangpeng

2017-07-12

As a typical example of large and complex mechanical systems, rotating machinery is prone to diversified sorts of mechanical faults. Among these faults, one of the prominent causes of malfunction is generated in gear transmission chains. Although they can be collected via vibration signals, the fault signatures are always submerged in overwhelming interfering contents. Therefore, identifying the critical fault's characteristic signal is far from an easy task. In order to improve the recognition accuracy of a fault's characteristic signal, a novel intelligent fault diagnosis method is presented. In this method, a dual-tree complex wavelet transform (DTCWT) is employed to acquire the multiscale signal's features. In addition, a convolutional neural network (CNN) approach is utilized to automatically recognise a fault feature from the multiscale signal features. The experiment results of the recognition for gear faults show the feasibility and effectiveness of the proposed method, especially in the gear's weak fault features.

High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) for Mass Spectrometry-Based Proteomics

PubMed Central

Swearingen, Kristian E.; Moritz, Robert L.

2013-01-01

SUMMARY High field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, we review recent developments in LC-FAIMS-MS and its application to MS-based proteomics. PMID:23194268

Mutations in GMPPB cause congenital myasthenic syndrome and bridge myasthenic disorders with dystroglycanopathies

PubMed Central

Belaya, Katsiaryna; Rodríguez Cruz, Pedro M.; Liu, Wei Wei; Maxwell, Susan; McGowan, Simon; Farrugia, Maria E.; Petty, Richard; Walls, Timothy J.; Sedghi, Maryam; Basiri, Keivan; Yue, Wyatt W.; Sarkozy, Anna; Bertoli, Marta; Pitt, Matthew; Kennett, Robin; Schaefer, Andrew; Bushby, Kate; Parton, Matt; Lochmüller, Hanns; Palace, Jacqueline; Muntoni, Francesco

2015-01-01

Congenital myasthenic syndromes are inherited disorders that arise from impaired signal transmission at the neuromuscular junction. Mutations in at least 20 genes are known to lead to the onset of these conditions. Four of these, ALG2, ALG14, DPAGT1 and GFPT1, are involved in glycosylation. Here we identify a fifth glycosylation gene, GMPPB, where mutations cause congenital myasthenic syndrome. First, we identified recessive mutations in seven cases from five kinships defined as congenital myasthenic syndrome using decrement of compound muscle action potentials on repetitive nerve stimulation on electromyography. The mutations were present through the length of the GMPPB, and segregation, in silico analysis, exon trapping, cell transfection followed by western blots and immunostaining were used to determine pathogenicity. GMPPB congenital myasthenic syndrome cases show clinical features characteristic of congenital myasthenic syndrome subtypes that are due to defective glycosylation, with variable weakness of proximal limb muscle groups while facial and eye muscles are largely spared. However, patients with GMPPB congenital myasthenic syndrome had more prominent myopathic features that were detectable on muscle biopsies, electromyography, muscle magnetic resonance imaging, and through elevated serum creatine kinase levels. Mutations in GMPPB have recently been reported to lead to the onset of muscular dystrophy dystroglycanopathy. Analysis of four additional GMPPB-associated muscular dystrophy dystroglycanopathy cases by electromyography found that a defective neuromuscular junction component is not always present. Thus, we find mutations in GMPPB can lead to a wide spectrum of clinical features where deficit in neuromuscular transmission is the major component in a subset of cases. Clinical recognition of GMPPB-associated congenital myasthenic syndrome may be complicated by the presence of myopathic features, but correct diagnosis is important because affected individuals can respond to appropriate treatments. PMID:26133662

A framework for evolutionary systems biology

PubMed Central

Loewe, Laurence

2009-01-01

Background Many difficult problems in evolutionary genomics are related to mutations that have weak effects on fitness, as the consequences of mutations with large effects are often simple to predict. Current systems biology has accumulated much data on mutations with large effects and can predict the properties of knockout mutants in some systems. However experimental methods are too insensitive to observe small effects. Results Here I propose a novel framework that brings together evolutionary theory and current systems biology approaches in order to quantify small effects of mutations and their epistatic interactions in silico. Central to this approach is the definition of fitness correlates that can be computed in some current systems biology models employing the rigorous algorithms that are at the core of much work in computational systems biology. The framework exploits synergies between the realism of such models and the need to understand real systems in evolutionary theory. This framework can address many longstanding topics in evolutionary biology by defining various 'levels' of the adaptive landscape. Addressed topics include the distribution of mutational effects on fitness, as well as the nature of advantageous mutations, epistasis and robustness. Combining corresponding parameter estimates with population genetics models raises the possibility of testing evolutionary hypotheses at a new level of realism. Conclusion EvoSysBio is expected to lead to a more detailed understanding of the fundamental principles of life by combining knowledge about well-known biological systems from several disciplines. This will benefit both evolutionary theory and current systems biology. Understanding robustness by analysing distributions of mutational effects and epistasis is pivotal for drug design, cancer research, responsible genetic engineering in synthetic biology and many other practical applications. PMID:19239699

Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

PubMed

Keerthana, S P; Kolandaivel, P

2015-04-01

Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

Recessive distal motor neuropathy with pyramidal signs in an Omani kindred: underlying novel mutation in the SIGMAR1 gene.

PubMed

Nandhagopal, R; Meftah, D; Al-Kalbani, S; Scott, P

2018-02-01

Distal hereditary motor neuropathy (dHMN) due to sigma non-opioid intracellular receptor 1 (SIGMAR1) gene mutation (OMIM 601978.0003) is a rare neuromuscular disorder characterized by prominent amyotrophic distal limb weakness and co-existing pyramidal signs initially described in a Chinese family recently. We report an extended consanguineous Omani family segregating dHMN with pyramidal signs in an autosomal recessive pattern and describe a novel mutation in the SIGMAR1 gene underlying this motor phenotype. We also provide an update on the reported phenotypic profile of SIGMAR1 mutations. We utilized homozygosity mapping and whole-exome sequencing of leucocyte DNA obtained from three affected members of an Omani family who manifested with a length-dependent motor neuropathy and pyramidal signs. We identified a novel C>T transition at nucleotide position 238 (c.238C>T) in exon 2 of the SIGMAR1 gene. Sanger sequencing and segregation analysis confirmed the presence of two copies of the variant in the affected subjects, unlike the unaffected healthy parents/sibling who carried, at most, a single copy. The T allele is predicted to cause a truncating mutation (p.Gln80*), probably flagging the mRNA for nonsense-mediated decay leading to a complete loss of function, thereby potentially contributing to the disease process. Our finding expands the spectrum of SIGMAR1 mutations causing recessive dHMN and indicates that this disorder is pan-ethnic. SIGMAR1 mutation should be included in the diagnostic panel of a dHMN, especially if there are co-existing pyramidal signs and autosomal recessive inheritance. © 2017 EAN.

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed Central

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-01-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

NASA Astrophysics Data System (ADS)

Faure, Guilhem; Koonin, Eugene V.

2015-05-01

Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores.

PubMed

Boyden, Steven E; Mahoney, Lane J; Kawahara, Genri; Myers, Jennifer A; Mitsuhashi, Satomi; Estrella, Elicia A; Duncan, Anna R; Dey, Friederike; DeChene, Elizabeth T; Blasko-Goehringer, Jessica M; Bönnemann, Carsten G; Darras, Basil T; Mendell, Jerry R; Lidov, Hart G W; Nishino, Ichizo; Beggs, Alan H; Kunkel, Louis M; Kang, Peter B

2012-05-01

We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism.

Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum.

PubMed

Ishikawa, Yoshihiro; Holden, Paul; Bächinger, Hans Peter

2017-10-20

Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/ SERPINH1 ) and 65-kDa FK506-binding protein (FKBP65/ FKBP10 ), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

PubMed

Lopez, G H; Morrison, J; Condon, J A; Wilson, B; Martin, J R; Liew, Y-W; Flower, R L; Hyland, C A

2015-10-01

Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status. © 2015 International Society of Blood Transfusion.

Modeling Interactions between Transposable Elements and the Plant Epigenetic Response: A Surprising Reliance on Element Retention.

PubMed

Roessler, Kyria; Bousios, Alexandros; Meca, Esteban; Gaut, Brandon S

2018-03-01

Transposable elements (TEs) compose the majority of angiosperm DNA. Plants counteract TE activity by silencing them epigenetically. One form of epigenetic silencing requires 21-22 nt small interfering RNAs that act to degrade TE mRNA and may also trigger DNA methylation. DNA methylation is reinforced by a second mechanism, the RNA-dependent DNA methylation (RdDM) pathway. RdDM relies on 24 nt small interfering RNAs and ultimately establishes TEs in a quiescent state. These host factors interact at a systems level, but there have been no system level analyses of their interactions. Here, we define a deterministic model that represents the propagation of active TEs, aspects of the host response and the accumulation of silenced TEs. We describe general properties of the model and also fit it to biological data in order to explore two questions. The first is why two overlapping pathways are maintained, given that both are likely energetically expensive. Under our model, RdDM silenced TEs effectively even when the initiation of silencing was weak. This relationship implies that only a small amount of RNAi is needed to initiate TE silencing, but reinforcement by RdDM is necessary to efficiently counter TE propagation. Second, we investigated the reliance of the host response on rates of TE deletion. The model predicted that low levels of deletion lead to few active TEs, suggesting that silencing is most efficient when methylated TEs are retained in the genome, thereby providing one explanation for the large size of plant genomes.

Experimental design to evaluate directed adaptive mutation in Mammalian cells.

PubMed

Bordonaro, Michael; Chiaro, Christopher R; May, Tobias

2014-12-09

We describe the experimental design for a methodological approach to determine whether directed adaptive mutation occurs in mammalian cells. Identification of directed adaptive mutation would have profound practical significance for a wide variety of biomedical problems, including disease development and resistance to treatment. In adaptive mutation, the genetic or epigenetic change is not random; instead, the presence and type of selection influences the frequency and character of the mutation event. Adaptive mutation can contribute to the evolution of microbial pathogenesis, cancer, and drug resistance, and may become a focus of novel therapeutic interventions. Our experimental approach was designed to distinguish between 3 types of mutation: (1) random mutations that are independent of selective pressure, (2) undirected adaptive mutations that arise when selective pressure induces a general increase in the mutation rate, and (3) directed adaptive mutations that arise when selective pressure induces targeted mutations that specifically influence the adaptive response. The purpose of this report is to introduce an experimental design and describe limited pilot experiment data (not to describe a complete set of experiments); hence, it is an early report. An experimental design based on immortalization of mouse embryonic fibroblast cells is presented that links clonal cell growth to reversal of an inactivating polyadenylation site mutation. Thus, cells exhibit growth only in the presence of both the countermutation and an inducing agent (doxycycline). The type and frequency of mutation in the presence or absence of doxycycline will be evaluated. Additional experimental approaches would determine whether the cells exhibit a generalized increase in mutation rate and/or whether the cells show altered expression of error-prone DNA polymerases or of mismatch repair proteins. We performed the initial stages of characterizing our system and have limited preliminary data from several pilot experiments. Cell growth and DNA sequence data indicate that we have identified a cell clone that exhibits several suitable characteristics, although further study is required to identify a more optimal cell clone. The experimental approach is based on a quantum biological model of basis-dependent selection describing a novel mechanism of adaptive mutation. This project is currently inactive due to lack of funding. However, consistent with the objective of early reports, we describe a proposed study that has not produced publishable results, but is worthy of report because of the hypothesis, experimental design, and protocols. We outline the project's rationale and experimental design, with its strengths and weaknesses, to stimulate discussion and analysis, and lay the foundation for future studies in this field.

Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury.

PubMed

Del Sorbo, Lorenzo; Costamagna, Andrea; Muraca, Giuseppe; Rotondo, Giuseppe; Civiletti, Federica; Vizio, Barbara; Bosco, Ornella; Martin Conte, Erica L; Frati, Giacomo; Delsedime, Luisa; Lupia, Enrico; Fanelli, Vito; Ranieri, V Marco

2016-08-01

Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation. Prospective, randomized, controlled experimental study. University research laboratory. C57/BL6 mice weighing 28-30 g. Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4°C), followed by 2 hours of warm (37°C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure. Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration. Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation.

Remarkable motor recovery after riboflavin therapy in adult-onset Brown—Vialetto—Van Laere syndrome

PubMed Central

Bashford, James A; Chowdhury, Fahmida A; Shaw, Chris E

2017-01-01

The clinical diagnosis of Brown—Vialetto—Van Laere syndrome in this woman with rapidly progressive pontobulbar palsy led to empirical high-dose oral riboflavin (1200 mg/day) therapy. This resulted in a dramatic improvement in her motor function from being anarthric, dysphagic, tetraparetic and in ventilatory failure to living independently with mild dysarthria and distal limb weakness. DNA sequencing of the SLC52A3 gene found compound heterozygous C-terminus mutations, V413A1/D461Y, consistent with recent reports of mutations within the riboflavin transporter genes (SLC52A2 and SLC52A3) in this condition. Early diagnosis and empirical riboflavin therapy can lead to major motor recovery in this condition, that can be sustained with long-term maintenance therapy. PMID:27777325

Molecular characterization of the Fy(a-b-) phenotype in a Polish family.

PubMed

Karolak, Ewa; Grodecka, Magdalena; Suchanowska, Anna; Klausa, Elżbieta; Bochenek, Stanisława; Majorczyk, Edyta; Czerwiński, Marcin; Waśniowska, Kazimiera

2013-10-01

The Fy(a-b-) phenotype, very rare in Caucasians and defined by the homozygous FY(*)B-33 allele, is associated with the -33T>C mutation in the promoter region of the FY gene. The allele FY(*)X is correlated with weak expression of Fy(b) antigen due to 265C>T and 298G>A mutations in FY(*)B allele. The purpose of this study was molecular characterization of Fy blood group antigens in Fy(a-b-) members of a Polish family. High-resolution melting analysis was performed to detect single nucleotide polymorphisms in amplified fragments of the FY gene. The Fy(a-b-) phenotype in three siblings of the Polish family was caused by the FY(*)X/FY(*)B-33 genotype. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sarcomeric gene mutations in sudden infant death syndrome (SIDS).

PubMed

Brion, Maria; Allegue, Catarina; Santori, Montserrat; Gil, Rocio; Blanco-Verea, Alejandro; Haas, Cordula; Bartsch, Christine; Poster, Simone; Madea, Burkhard; Campuzano, Oscar; Brugada, Ramon; Carracedo, Angel

2012-06-10

In developed countries, sudden infant death syndrome (SIDS) represents the most prevalent cause of death in children between 1 month and 1 year of age. SIDS is a diagnosis of exclusion, a negative autopsy which requires the absence of structural organ disease. Although investigators have confirmed that a significant percentage of SIDS cases are actually channelopathies, no data have been made available as to whether other sudden cardiac death-associated diseases, such as hypertrophic cardiomyopathy (HCM), could be responsible for some cases of SIDS. The presence of a genetic mutation in the sarcomeric protein usually affects the force of contraction of the myocyte, whose weakness is compensated with progressive hypertrophy and disarray. However, it is unclear whether in the most incipient forms, that is, first years of life, the lack of these phenotypes still confers a risk of arrhythmogenesis. The main goal of the present study is to wonder whether genetic defects in the sarcomeric proteins, previously associated with HCM, could be responsible for SIDS. We have analysed 286 SIDS cases for the most common genes implicated in HCM in adults. A total of 680 mutations localised in 16 genes were analysed by semi-automated matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDITOF-MS) using the Sequenom MassARRAY(®) System. Ten subjects with completely normal hearts showed mutated alleles at nine of the genetic variants analysed, and one additional novel mutation was detected by conventional sequencing. Therefore, a genetic mutation associated with HCM may cause sudden cardiac death in the absence of an identifiable phenotype. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Gas Chromatographic/Mass Spectrometric Differentiation of Atenolol, Metoprolol, Propranolol, and an Interfering Metabolite Product of Metoprolol

DTIC Science & Technology

2004-10-01

Gas Chromatographic/Mass Spectrometric Differentiation of Atenolol, Metoprolol , Propranolol, and an Interfering Metabolite Product of Metoprolol ...4. Title and Subtitle 5. Report Date October 2004 Gas Chromatographic/Mass Spectrometric Differentiation of Atenolol, Metoprolol , Propranolol...and an Interfering Metabolite Product of Metoprolol 6. Performing Organization Code 7. Author(s) 8. Performing Organization Report No. Angier MK

Late-onset dysferlinopathy presented as "liver enzyme" abnormalities: a technical note.

PubMed

Li, Fang; Yin, Geng; Xie, Qibing; Shi, Guixiu

2014-08-01

Limb-girdle muscular dystrophy type 2B,a type of dysferlinopathy, is caused by mutations in the dysferlin gene (DYSE). It is characterized by predominant weakness and atrophy of muscles of the pelvic and shoulder girdles, massive elevation of serum CK concentration, slow progression, and onset usually within the second or third decade of life. We present a Chinese patient whose disease onset was at the age of 50 years with persistent elevation of transaminases for 3 years before weakness appeared. She had been considered as having liver disease for a long time and then polymyositis. Finally, biceps brachii biopsy revealed dystrophic morphology and depletion of dysferlin in immunohistochemistry. This case should remind readers that late-age onset of dysferlinopathy can be misdiagnosed as liver disease or polymyositis.

[The influence of interfered circadian rhythm on pregnancy and neonatal rats].

PubMed

Chen, Wen-Jun; Sheng, Wen-Jie; Guo, Yin-Hua; Tan, Yong

2015-10-25

The aim of this study was to observe the influence of interfered circadian rhythm on pregnancy of rats and growth of neonatal rats, and to explore the relationship between the interfered circadian rhythm and the changes of melatonin and progesterone. Continuous light was used to inhibit melatonin secretion and therefore the interfered circadian rhythm animal model was obtained. The influence of interfered circadian rhythm on delivery of pregnant rats was observed. Serum was collected from rats during different stages of pregnancy to measure the concentrations of melatonin and progesterone. In order to observe the embryo resorption rate, half of pregnant rats were randomly selected to undergo a laparotomy, and the remainder was used to observe delivery and assess the growth of neonatal rats after delivery. The results showed that the interfered circadian rhythm induced adverse effects on pregnancy outcomes, including an increase of embryo resorption rate and a decrease in the number of live births; inhibited the secretion of melatonin along with decreased serum progesterone level; prolonged the stage of labor, but not the duration of pregnancy; and disturbed the fetal intrauterine growth and the growth of neonatal rats. The results suggest that interfered circadian rhythm condition made by continuous light could make adverse effects on both pregnant rats and neonatal rats. The results of our study may provide a way to modulate pregnant women's circadian rhythm and a possibility of application of melatonin on pregnant women.

NaV1.4 mutations cause hypokalaemic periodic paralysis by disrupting IIIS4 movement during recovery

PubMed Central

Lehmann-Horn, Frank; Fan, Chunxiang; Wolf, Markus; Winston, Vern; Merlini, Luciano

2014-01-01

Hypokalaemic periodic paralysis is typically associated with mutations of voltage sensor residues in calcium or sodium channels of skeletal muscle. To date, causative sodium channel mutations have been studied only for the two outermost arginine residues in S4 voltage sensor segments of domains I to III. These mutations produce depolarization of skeletal muscle fibres in response to reduced extracellular potassium, owing to an inward cation-selective gating pore current activated by hyperpolarization. Here, we describe mutations of the third arginine, R3, in the domain III voltage sensor i.e. an R1135H mutation which was found in two patients in separate families and a novel R1135C mutation identified in a third patient in another family. Muscle fibres from a patient harbouring the R1135H mutation showed increased depolarization tendency at normal and reduced extracellular potassium compatible with the diagnosis. Additionally, amplitude and rise time of action potentials were reduced compared with controls, even for holding potentials at which all NaV1.4 are fully recovered from inactivation. These findings may be because of an outward omega current activated at positive potentials. Expression of R1135H/C in mammalian cells indicates further gating defects that include significantly enhanced entry into inactivation and prolonged recovery that may additionally contribute to action potential inhibition at the physiological resting potential. After S4 immobilization in the outward position, mutant channels produce an inward omega current that most likely depolarizes the resting potential and produces the hypokalaemia-induced weakness. Gating current recordings reveal that mutations at R3 inhibit S4 deactivation before recovery, and molecular dynamics simulations suggest that this defect is caused by disrupted interactions of domain III S2 countercharges with S4 arginines R2 to R4 during repolarization of the membrane. This work reveals a novel mechanism of disrupted S4 translocation for hypokalaemic periodic paralysis mutations at arginine residues located below the gating pore constriction of the voltage sensor module. PMID:24549961

A NOVEL MUTATION IN THE HCN4 GENE CAUSES SYMPTOMATIC SINUS BRADYCARDIA IN MOROCCAN JEWS

PubMed Central

Laish-Farkash, Avishag; Brass, Dovrat; Marek-Yagel, Dina; Pras, Elon; Dascal, Nathan; Antzelevitch, Charles; Nof, Eyal; Reznik, Haya; Eldar, Michael; Glikson, Michael; Luria, David

2010-01-01

Objectives To conduct a clinical, genetic and functional analysis of three unrelated families with familial sinus bradycardia (FSB). Background Mutations in the hyperpolarization-activated nucleotide-gated channel (HCN4) are known to be associated with FSB. Methods and Results Three males of Moroccan Jewish descent were hospitalized: one survived an out-of-hospital cardiac arrest and 2 presented with weakness and presyncopal events. All 3 had significant sinus bradycardia, also found in other first-degree relatives, with a segregation suggesting autosomal-dominant inheritance. All had normal response to exercise and normal heart structure. Sequencing of the HCN4 gene in all patients revealed a C to T transition at nucleotide position 1454, which resulted in an alanine to valine change (A485V) in the ion channel pore found in most of their bradycardiac relatives, but not in 150 controls. Functional expression of the mutated ion channel in Xenopus oocytes and in human embryonic kidney 293 cells revealed profoundly reduced function and synthesis of the mutant channel compared to wild-type. Conclusions We describe a new mutation in the HCN4 gene causing symptomatic FSB in 3 unrelated individuals of similar ethnic background that may indicate unexplained FSB in this ethnic group. This profound functional defect is consistent with the symptomatic phenotype. PMID:20662977

Myasthenic syndromes due to defects in COL13A1 and in the N-linked glycosylation pathway.

PubMed

Beeson, David; Cossins, Judith; Rodriguez-Cruz, Pedro; Maxwell, Susan; Liu, Wei-Wei; Palace, Jacqueline

2018-02-01

The congenital myasthenic syndromes (CMS) are hereditary disorders of neuromuscular transmission. The number of cases recognized, at around 1:100,000 in the United Kingdom, is increasing with improved diagnosis. The advent of next-generation sequencing has facilitated the discovery of many genes that harbor CMS-associated mutations. An emerging group of CMS, characterized by a limb-girdle pattern of muscle weakness, is caused by mutations in genes that encode proteins involved in the initial steps of the N-linked glycosylation pathway, which is surprising, since this pathway is found in all mammalian cells. However, mutations in these genes may also give rise to multisystem disorders (congenital disorders of glycosylation) or muscle disorders where the myasthenic symptoms constitute only one component within a wider phenotypic spectrum. We also report a CMS due to mutations in COL13A1, which encodes an extracellular matrix protein that is concentrated at the neuromuscular junction and highlights a role for these extracellular matrix proteins in maintaining synaptic stability that is independent of the AGRN/MuSK clustering pathway. Knowledge about the neuromuscular synapse and the different proteins involved in maintaining its structure as well as function enables us to tailor treatments to the underlying pathogenic mechanisms. © 2018 New York Academy of Sciences.

Charcot-Marie-Tooth disease type 2 caused by homozygous MME gene mutation superimposed by chronic inflammatory demyelinating polyneuropathy.

PubMed

Fujisawa, Miwako; Sano, Yasuteru; Omoto, Masatoshi; Ogasawara, Jyun-Ichi; Koga, Michiaki; Takashima, Hiroshi; Kanda, Takashi

2017-09-30

We report a 59-year-old Japanese male who developed gradually worsening weakness and numbness of distal four extremities since age 50. His parents were first cousins, and blood and cerebral spinal examinations were unremarkable. Homozygous mutation of MME gene was detected and thus he was diagnosed as autosomal-recessive Charcot-Marie-Tooth disease 2T (AR-CMT2T); however, electrophysiological examinations revealed scattered demyelinative changes including elongated terminal latency in several peripheral nerve trunks. Sural nerve biopsy showed endoneurial edema and a lot of thinly myelinated nerve fibers with uneven distribution of remnant myelinated fibers within and between fascicles. Immunoglobulin treatment was initiated considering the possibility of superimposed inflammation and demyelination, and immediate clinical as well as electrophysiological improvements were noted. Our findings indicate that AR-CMT2T caused by MME mutation predisposes to a superimposed inflammatory demyelinating neuropathy. This is the first report which documented the co-existence of CMT2 and chronic inflammatory demyelinating polyneuropathy (CIDP); however, in the peripheral nervous system, neprilysin, a product of MME gene, is more abundant in myelin sheath than in axonal component. The fragility of myelin sheath due to mutated neprilysin may trigger the detrimental immune response against peripheral myelin in this patient.

Genetic control of enhanced mutability of mitochondrial DNA and gamma-ray sensitivity in Saccharomyces cerevisiae.

PubMed Central

Foury, F; Goffeau, A

1979-01-01

Five nuclear mutants enhancing the spontaneous mutation rate of mtDNA have been isolated in Saccharomyces cerevisiae. These mutators fall into five complementation groups and are located at five genetic loci different from rad50 to rad57 loci. Three mutants (gam1, gam2, and gam4), insensitive or weakly sensitive to gamma-rays, exhibit increased frequency of spontaneous production of mutants with large deletions of the mtDNA (p-) and of all tested mitochondrial drug-resistant mutants. Two other mutants (gam3 and gam5), highly sensitive to gamma-rays, increase only the mutation rate of particular alleles of the mtDNA. The mutant gam5 enhances only the production of p- and erythromycin-resistant clones. The mutant gam3 exhibits an enhanced rate of oligomycin-resistant clones as well as a collateral increase of nuclear mutability. The existence of gam3 and gam5 mutants indicates that at least two common steps control both nuclear DNA repair and the mutability of particular alleles of the mtDNA. However, the general spontaneous mutability of the mtDNA includes at least three steps not involved in the repair of nuclear DNA, as revealed by the gam1, gam2, and gam4 mutations. PMID:392521

Late onset of neutral lipid storage disease due to novel PNPLA2 mutations causing total loss of lipase activity in a patient with myopathy and slight cardiac involvement.

PubMed

Missaglia, Sara; Maggi, Lorenzo; Mora, Marina; Gibertini, Sara; Blasevich, Flavia; Agostoni, Piergiuseppe; Moro, Laura; Cassandrini, Denise; Santorelli, Filippo Maria; Gerevini, Simonetta; Tavian, Daniela

2017-05-01

Neutral lipid storage disease with myopathy (NLSDM) presents with skeletal muscle myopathy and severe dilated cardiomyopathy in nearly 40% of cases. NLSDM is caused by mutations in the PNPLA2 gene, which encodes the adipose triglyceride lipase (ATGL). Here we report clinical and genetic findings of a patient carrying two novel PNPLA2 mutations (c.696+4A>G and c.553_565delGTCCCCCTTCTCG). She presented at age 39 with right upper limb abduction weakness slowly progressing over the years with asymmetric involvement of proximal upper and lower limb muscles. Cardiological evaluation through ECG and heart echo scan was normal until the age 53, when mild left ventricular diastolic dysfunction was detected. Molecular analysis revealed that only one type of PNPLA2 transcript, with exon 5 skipping, was expressed in patient cells. Such aberrant mRNA causes the production of a shorter ATGL protein, lacking part of the catalytic domain. This is an intriguing case, displaying severe PNPLA2 mutations with clinical presentation characterized by slight cardiac impairment and full expression of severe asymmetric myopathy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Early-progressive dilated cardiomyopathy in a family with Becker muscular dystrophy related to a novel frameshift mutation in the dystrophin gene exon 27.

PubMed

Tsuda, Takeshi; Fitzgerald, Kristi; Scavena, Mena; Gidding, Samuel; Cox, Mary O; Marks, Harold; Flanigan, Kevin M; Moore, Steven A

2015-03-01

We report a family in which two male siblings with Becker muscular dystrophy (BMD) developed severe dilated cardiomyopathy (DCM) and progressive heart failure (HF) at age 11 years; one died at age 14 years while awaiting heart transplant and the other underwent left ventricular assist device implantation at the same age. Genetic analysis of one sibling showed a novel frameshift mutation in exon 27 of Duchenne muscular dystrophy (DMD) gene (c.3779_3785delCTTTGGAinsGG), in which seven base pairs are deleted and two are inserted. Although this predicts an amino-acid substitution and premature termination (p.Thr1260Argfs*8), muscle biopsy dystrophin immunostaining instead indicates that the mutation is more likely to alter splicing. Despite relatively preserved skeletal muscular performance, both the siblings developed progressive HF secondary to early-onset DCM. In addition, their 7-year-old nephew with delayed gross motor development, mild proximal muscle weakness and markedly elevated serum creatine kinase level (>13 000 IU l(-1)) at 16 months was recently demonstrated to have the familial DMD mutation. Here, we report a novel genotype of BMD with early-onset DCM and progressive lethal HF during early adolescence.

A de novo mutation of the MYH7 gene in a large Chinese family with autosomal dominant myopathy

PubMed Central

Oda, Tetsuya; Xiong, Hui; Kobayashi, Kazuhiro; Wang, Shuo; Satake, Wataru; Jiao, Hui; Yang, Yanling; Cha, Pei-Chieng; Hayashi, Yukiko K; Nishino, Ichizo; Suzuki, Yutaka; Sugano, Sumio; Wu, Xiru; Toda, Tatsushi

2015-01-01

Laing distal myopathy (LDM) is an autosomal dominant myopathy that is caused by mutations in the slow/beta cardiac myosin heavy-chain (MYH7) gene. It has been recently reported that LDM presents with a wide range of clinical manifestations. We herein report a large Chinese family with autosomal dominant myopathy. The affected individuals in the family presented with foot drop in early childhood, along with progressive distal and proximal limb weakness. Their characteristic symptoms include scapular winging and scoliosis in the early disease phase and impairment of ambulation in the advanced phase. Although limb-girdle muscle dystrophy (LGMD) was suspected initially, a definite diagnosis could not be reached. As such, we performed linkage analysis and detected four linkage regions, namely 1q23.2-24.1, 14q11.2-12, 15q26.2-26.3 and 17q24.3. Through subsequent whole exome sequencing, we found a de novo p.K1617del causative mutation in the MYH7 gene and diagnosed the disease as LDM. This is the first LDM case in China. Our patients have severe clinical manifestations that mimic LGMD in comparison with the patients with the same mutation reported elsewhere. PMID:27081534

Both Epistasis and Diversifying Selection Drive the Structural Evolution of the Ebola Virus Glycoprotein Mucin-Like Domain.

PubMed

Ibeh, Neke; Nshogozabahizi, Jean Claude; Aris-Brosou, Stéphane

2016-06-01

Throughout the last 3 decades, Ebola virus (EBOV) outbreaks have been confined to isolated areas within Central Africa; however, the 2014 variant reached unprecedented transmission and mortality rates. While the outbreak was still under way, it was reported that the variant leading up to this outbreak evolved faster than previous EBOV variants, but evidence for diversifying selection was undetermined. Here, we test this selection hypothesis and show that while previous EBOV outbreaks were preceded by bursts of diversification, evidence for site-specific diversifying selection during the emergence of the 2014 EBOV clade is weak. However, we show strong evidence supporting an interplay between selection and correlated evolution (epistasis), particularly in the mucin-like domain (MLD) of the EBOV glycoprotein. By reconstructing ancestral structures of the MLD, we further propose a structural mechanism explaining how the substitutions that accumulated between 1918 and 1969 distorted the MLD, while more recent epistatic substitutions restored part of the structure, with the most recent substitution being adaptive. We suggest that it is this complex interplay between weak selection, epistasis, and structural constraints that has shaped the evolution of the 2014 EBOV variant. The role that selection plays in the emergence of viral epidemics remains debated, particularly in the context of the 2014 EBOV outbreak. Most critically, should such evidence exist, it is generally unclear how this relates to function and increased virulence. Here, we show that the viral lineage leading up to the 2014 outbreak underwent a complex interplay between selection and correlated evolution (epistasis) in a protein region that is critical for immune evasion. We then reconstructed the three-dimensional structure of this domain and showed that the initial mutations in this lineage deformed the structure, while subsequent mutations restored part of the structure. Along this mutational path, the first and last mutations were adaptive, while the intervening ones were epistatic. Altogether, we provide a mechanistic model that explains how selection and epistasis acted on the structural constraints that materialized during the 2014 EBOV outbreak. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

p53 expression and mutation analysis of odontogenic cysts with and without dysplasia.

PubMed

Cox, Darren P

2012-01-01

Overexpression of p53 protein is well described in odontogenic cystic lesions (OCLs), including those with epithelial dysplasia; however, most p53 antibodies stain both wild-type and mutated p53 protein and may not reflect genotype. Direct sequencing of the p53 gene has not identified mutations in OCLs with dysplasia. The purpose of this study was to determine the molecular basis of p53 expression in several types of OCLs with and without dysplasia. The study material comprised 13 OCLs: odontogenic keratocyst (n = 5), orthokeratinized odontogenic cyst (n = 5), dentigerous cyst (n = 2), lateral periodontal cyst (n = 1), and unspecified developmental odontogenic cyst (UDOC) (n = 1). Five of these had features of mild or moderate epithelial dysplasia. One intraosseous squamous cell carcinoma (SCC) that was believed to have arisen from an antecedent dysplastic orthokeratinized OC was also included. Immunohistochemistry was performed using the DO7 monoclonal antibody that recognizes wild-type and mutated p53. DNA was extracted from microdissected tissue for all samples and exons 4 to 8 of the p53 gene direct sequenced. In 4 of 5 OCLs with dysplasia there was strong nuclear staining of basal and suprabasal cells. In all cases without dysplasia, nuclear expression in basal cells was either negative or weak and was absent in suprabasal cell nuclei. A mutation in exon 6 of the p53 gene (E224D) was identified in both the dysplastic orthokeratinized OC and the subsequent intraosseous SCC. OCLs with features of dysplasia show increased expression of p53 protein that does not reflect p53 mutational status. One dysplastic OC shared the same p53 mutation with a subsequent intraosseous SCC, indicating that p53 mutation may be associated with malignant transformation in this case. Copyright © 2012 Elsevier Inc. All rights reserved.

Spinal Muscular Atrophy With Respiratory Distress Type 1-A Child With Atypical Presentation.

PubMed

Chiu, Annie Ting Gee; Chan, Sophelia Hoi Shan; Wu, Shun Ping; Ting, Shun Hin; Chung, Brian Hon Yin; Chan, Angel On Kei; Wong, Virginia Chun Nei

2018-01-01

The authors report a child with spinal muscular atrophy with respiratory distress type 1 (SMARD1). She presented atypically with hypothyroidism and heart failure due to septal defects that required early heart surgery and microcephaly in association with cerebral atrophy and thin corpus collosum. The subsequent asymmetrical onset of diaphragmatic paralysis, persistent hypotonia, and generalized muscle weakness led to the suspicion of spinal muscular atrophy with respiratory distress type 1. Sanger sequencing confirmed a compound heterozygous mutation in the Immunoglobulin Mu Binding Protein 2 (IGHMBP2) gene, with a known mutation c.2362C > T (p.Arg788*) and a novel frameshift mutation c.2048delG (p.Gly683A1afs*50). Serial nerve conduction study and electromyography confirmed progressive sensorimotor polyneuropathy and neuronopathy. In summary, this case report describes a child with spinal muscular atrophy with respiratory distress type 1 also with congenital cardiac disease and endocrine dysfunction, expanding the phenotypic spectrum of this condition. A high index of suspicion is needed in diagnosing this rare condition to guide the management and genetic counseling.

A novel homozygous Fas ligand mutation leads to early protein truncation, abrogation of death receptor and reverse signaling and a severe form of the autoimmune lymphoproliferative syndrome.

PubMed

Nabhani, Schafiq; Hönscheid, Andrea; Oommen, Prasad T; Fleckenstein, Bernhard; Schaper, Jörg; Kuhlen, Michaela; Laws, Hans-Jürgen; Borkhardt, Arndt; Fischer, Ute

2014-12-01

We report a novel type of mutation in the death ligand FasL that was associated with a severe phenotype of the autoimmune lymphoproliferative syndrome in two patients. A frameshift mutation in the intracellular domain led to complete loss of FasL expression. Cell death signaling via its receptor and reverse signaling via its intracellular domain were completely abrogated. In vitro lymphocyte proliferation induced by weak T cell receptor stimulation could be blocked and cell death was induced by engagement of FasL in T cells derived from healthy individuals and a heterozygous carrier, but not in FasL-deficient patient derived cells. Expression of genes implicated in lymphocyte proliferation and activation (CCND1, NFATc1, NF-κB1) was increased in FasL-deficient T cells and could not be downregulated by FasL engagement as in healthy cells. Our data thus suggest, that deficiency in FasL reverse signaling may contribute to the clinical lymphoproliferative phenotype of ALPS. Copyright © 2014 Elsevier Inc. All rights reserved.

A Case of Brown-Vialetto-Van Laere Syndrome Due To a Novel Mutation in SLC52A3 Gene

PubMed Central

Thulasi, Venkatraman; Veerapandiyan, Aravindhan; Pletcher, Beth A.; Tong, Chun M.

2017-01-01

Brown-Vialetto-Van Laere syndrome is a rare disorder characterized by motor, sensory, and cranial neuronopathies, associated with mutations in SLC52A2 and SLC52A3 genes that code for human riboflavin transporters RFVT2 and RFVT3, respectively. The authors describe the clinical course of a 6-year-old girl with Brown-Vialetto-Van Laere syndrome and a novel homozygous mutation c.1156T>C in the SLC52A3 gene, who presented at the age of 2.5 years with progressive brain stem dysfunction including ptosis, facial weakness, hearing loss, dysphagia, anarthria with bilateral vocal cord paralysis, and ataxic gait. She subsequently developed respiratory failure requiring tracheostomy and worsening dysphagia necessitating a gastrostomy. Following riboflavin supplementation, resolution of facial diplegia and ataxia, improvements in ptosis, and bulbar function including vocalization and respiration were noted. However, her sensorineural hearing loss remained unchanged. Similar to other cases of Brown-Vialetto-Van Laere syndrome, our patient responded favorably to early riboflavin supplementation with significant but not complete neurologic recovery. PMID:28856173

A Case of Brown-Vialetto-Van Laere Syndrome Due To a Novel Mutation in SLC52A3 Gene: Clinical Course and Response to Riboflavin.

PubMed

Thulasi, Venkatraman; Veerapandiyan, Aravindhan; Pletcher, Beth A; Tong, Chun M; Ming, Xue

2017-01-01

Brown-Vialetto-Van Laere syndrome is a rare disorder characterized by motor, sensory, and cranial neuronopathies, associated with mutations in SLC52A2 and SLC52A3 genes that code for human riboflavin transporters RFVT2 and RFVT3, respectively. The authors describe the clinical course of a 6-year-old girl with Brown-Vialetto-Van Laere syndrome and a novel homozygous mutation c.1156T>C in the SLC52A3 gene, who presented at the age of 2.5 years with progressive brain stem dysfunction including ptosis, facial weakness, hearing loss, dysphagia, anarthria with bilateral vocal cord paralysis, and ataxic gait. She subsequently developed respiratory failure requiring tracheostomy and worsening dysphagia necessitating a gastrostomy. Following riboflavin supplementation, resolution of facial diplegia and ataxia, improvements in ptosis, and bulbar function including vocalization and respiration were noted. However, her sensorineural hearing loss remained unchanged. Similar to other cases of Brown-Vialetto-Van Laere syndrome, our patient responded favorably to early riboflavin supplementation with significant but not complete neurologic recovery.

Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells.

PubMed

Söllner, Christian; Rauch, Gerd-Jörg; Siemens, Jan; Geisler, Robert; Schuster, Stephan C; Müller, Ulrich; Nicolson, Teresa

2004-04-29

Hair cells have highly organized bundles of apical projections, or stereocilia, that are deflected by sound and movement. Displacement of stereocilia stretches linkages at the tips of stereocilia that are thought to gate mechanosensory channels. To identify the molecular machinery that mediates mechanotransduction in hair cells, zebrafish mutants were identified with defects in balance and hearing. In sputnik mutants, stereociliary bundles are splayed to various degrees, with individuals displaying reduced or absent mechanotransduction. Here we show that the defects in sputnik mutants are caused by mutations in cadherin 23 (cdh23). Mutations in Cdh23 also cause deafness and vestibular defects in mice and humans, and the protein is present in hair bundles. We show that zebrafish Cdh23 protein is concentrated near the tips of hair bundles, and that tip links are absent in homozygous sputnik(tc317e) larvae. Moreover, tip links are absent in larvae carrying weak alleles of cdh23 that affect mechanotransduction but not hair bundle integrity. We conclude that Cdh23 is an essential tip link component required for hair-cell mechanotransduction.

Formal properties of the probability of fixation: identities, inequalities and approximations.

PubMed

McCandlish, David M; Epstein, Charles L; Plotkin, Joshua B

2015-02-01

The formula for the probability of fixation of a new mutation is widely used in theoretical population genetics and molecular evolution. Here we derive a series of identities, inequalities and approximations for the exact probability of fixation of a new mutation under the Moran process (equivalent results hold for the approximate probability of fixation under the Wright-Fisher process, after an appropriate change of variables). We show that the logarithm of the fixation probability has particularly simple behavior when the selection coefficient is measured as a difference of Malthusian fitnesses, and we exploit this simplicity to derive inequalities and approximations. We also present a comprehensive comparison of both existing and new approximations for the fixation probability, highlighting those approximations that induce a reversible Markov chain when used to describe the dynamics of evolution under weak mutation. To demonstrate the power of these results, we consider the classical problem of determining the total substitution rate across an ensemble of biallelic loci and prove that, at equilibrium, a strict majority of substitutions are due to drift rather than selection. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluating the evaluation of cancer driver genes

PubMed Central

Tokheim, Collin J.; Papadopoulos, Nickolas; Kinzler, Kenneth W.; Vogelstein, Bert; Karchin, Rachel

2016-01-01

Sequencing has identified millions of somatic mutations in human cancers, but distinguishing cancer driver genes remains a major challenge. Numerous methods have been developed to identify driver genes, but evaluation of the performance of these methods is hindered by the lack of a gold standard, that is, bona fide driver gene mutations. Here, we establish an evaluation framework that can be applied to driver gene prediction methods. We used this framework to compare the performance of eight such methods. One of these methods, described here, incorporated a machine-learning–based ratiometric approach. We show that the driver genes predicted by each of the eight methods vary widely. Moreover, the P values reported by several of the methods were inconsistent with the uniform values expected, thus calling into question the assumptions that were used to generate them. Finally, we evaluated the potential effects of unexplained variability in mutation rates on false-positive driver gene predictions. Our analysis points to the strengths and weaknesses of each of the currently available methods and offers guidance for improving them in the future. PMID:27911828

Clinical heterogeneity and phenotype/genotype findings in 5 families with GYG1 deficiency

PubMed Central

Ben Yaou, Rabah; Hubert, Aurélie; Nelson, Isabelle; Dahlqvist, Julia R.; Gaist, David; Streichenberger, Nathalie; Beuvin, Maud; Krahn, Martin; Petiot, Philippe; Parisot, Frédéric; Michel, Fabrice; Malfatti, Edoardo; Romero, Norma; Carlier, Robert Yves; Eymard, Bruno; Labrune, Philippe; Duno, Morten; Krag, Thomas; Cerino, Mathieu; Bartoli, Marc; Bonne, Gisèle; Vissing, John; Laforet, Pascal

2017-01-01

Objective: To describe the variability of muscle symptoms in patients carrying mutations in the GYG1 gene, encoding glycogenin-1, an enzyme involved in the biosynthesis of glycogen, and to discuss genotype-phenotype relations. Methods: We describe 9 patients from 5 families in whom muscle biopsies showed vacuoles with an abnormal accumulation of glycogen in muscle fibers, partially α-amylase resistant suggesting polyglucosan bodies. The patients had either progressive early-onset limb-girdle weakness or late-onset distal or scapuloperoneal muscle affection as shown by muscle imaging. No clear definite cardiac disease was found. Histologic and protein analysis investigations were performed on muscle. Results: Genetic analyses by direct or exome sequencing of the GYG1 gene revealed 6 different GYG1 mutations. Four of the mutations were novel. They were compound heterozygous in 3 families and homozygous in 2. Protein analysis revealed either the absence of glycogenin-1 or reduced glycogenin-1 expression with impaired glucosylation. Conclusions: Our report extends the genetic and clinical spectrum of glycogenin-1–related myopathies to include scapuloperoneal and distal affection with glycogen accumulation. PMID:29264399

Clinical heterogeneity and phenotype/genotype findings in 5 families with GYG1 deficiency.

PubMed

Ben Yaou, Rabah; Hubert, Aurélie; Nelson, Isabelle; Dahlqvist, Julia R; Gaist, David; Streichenberger, Nathalie; Beuvin, Maud; Krahn, Martin; Petiot, Philippe; Parisot, Frédéric; Michel, Fabrice; Malfatti, Edoardo; Romero, Norma; Carlier, Robert Yves; Eymard, Bruno; Labrune, Philippe; Duno, Morten; Krag, Thomas; Cerino, Mathieu; Bartoli, Marc; Bonne, Gisèle; Vissing, John; Laforet, Pascal; Petit, François M

2017-12-01

To describe the variability of muscle symptoms in patients carrying mutations in the GYG1 gene, encoding glycogenin-1, an enzyme involved in the biosynthesis of glycogen, and to discuss genotype-phenotype relations. We describe 9 patients from 5 families in whom muscle biopsies showed vacuoles with an abnormal accumulation of glycogen in muscle fibers, partially α-amylase resistant suggesting polyglucosan bodies. The patients had either progressive early-onset limb-girdle weakness or late-onset distal or scapuloperoneal muscle affection as shown by muscle imaging. No clear definite cardiac disease was found. Histologic and protein analysis investigations were performed on muscle. Genetic analyses by direct or exome sequencing of the GYG1 gene revealed 6 different GYG1 mutations. Four of the mutations were novel. They were compound heterozygous in 3 families and homozygous in 2. Protein analysis revealed either the absence of glycogenin-1 or reduced glycogenin-1 expression with impaired glucosylation. Our report extends the genetic and clinical spectrum of glycogenin-1-related myopathies to include scapuloperoneal and distal affection with glycogen accumulation.

Potassium dependent rescue of a myopathy with core-like structures in mouse

PubMed Central

Hanson, M Gartz; Wilde, Jonathan J; Moreno, Rosa L; Minic, Angela D; Niswander, Lee

2015-01-01

Myopathies decrease muscle functionality. Mutations in ryanodine receptor 1 (RyR1) are often associated with myopathies with microscopic core-like structures in the muscle fiber. In this study, we identify a mouse RyR1 model in which heterozygous animals display clinical and pathological hallmarks of myopathy with core-like structures. The RyR1 mutation decreases sensitivity to activated calcium release and myoplasmic calcium levels, subsequently affecting mitochondrial calcium and ATP production. Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis. Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology. We identify regulators of potassium homeostasis as biomarkers of disease that may reveal therapeutic targets in human patients with myopathy of central core disease (CCD). Altogether, our results suggest that amelioration of potassium leaks through potassium homeostasis mechanisms may minimize muscle damage of myopathies due to certain RyR1 mutations. DOI: http://dx.doi.org/10.7554/eLife.02923.001 PMID:25564733

A diffusion approach to approximating preservation probabilities for gene duplicates.

PubMed

O'Hely, Martin

2006-08-01

Consider a haploid population and, within its genome, a gene whose presence is vital for the survival of any individual. Each copy of this gene is subject to mutations which destroy its function. Suppose one member of the population somehow acquires a duplicate copy of the gene, where the duplicate is fully linked to the original gene's locus. Preservation is said to occur if eventually the entire population consists of individuals descended from this one which initially carried the duplicate. The system is modelled by a finite state-space Markov process which in turn is approximated by a diffusion process, whence an explicit expression for the probability of preservation is derived. The event of preservation can be compared to the fixation of a selectively neutral gene variant initially present in a single individual, the probability of which is the reciprocal of the population size. For very weak mutation, this and the probability of preservation are equal, while as mutation becomes stronger, the preservation probability tends to double this reciprocal. This is in excellent agreement with simulation studies.

Proactive routing mutation against stealthy Distributed Denial of Service attacks: metrics, modeling, and analysis

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

Duan, Qi; Al-Shaer, Ehab; Chatterjee, Samrat

The Infrastructure Distributed Denial of Service (IDDoS) attacks continue to be one of the most devastating challenges facing cyber systems. The new generation of IDDoS attacks exploit the inherent weakness of cyber infrastructure including deterministic nature of routes, skew distribution of flows, and Internet ossification to discover the network critical links and launch highly stealthy flooding attacks that are not observable at the victim end. In this paper, first, we propose a new metric to quantitatively measure the potential susceptibility of any arbitrary target server or domain to stealthy IDDoS attacks, and es- timate the impact of such susceptibility onmore » enterprises. Second, we develop a proactive route mutation technique to minimize the susceptibility to these attacks by dynamically changing the flow paths periodically to invalidate the adversary knowledge about the network and avoid targeted critical links. Our proposed approach actively changes these network paths while satisfying security and qualify of service requirements. We present an integrated approach of proactive route mutation that combines both infrastructure-based mutation that is based on reconfiguration of switches and routers, and middle-box approach that uses an overlay of end-point proxies to construct a virtual network path free of critical links to reach a destination. We implemented the proactive path mutation technique on a Software Defined Network using the OpendDaylight controller to demonstrate a feasible deployment of this approach. Our evaluation validates the correctness, effectiveness, and scalability of the proposed approaches.« less

SDN-1/syndecan regulates growth factor signaling in distal tip cell migrations in C. elegans.

PubMed

Schwabiuk, Megan; Coudiere, Ludivine; Merz, David C

2009-10-01

Mutations in the sdn-1/syndecan gene act as genetic enhancers of the ventral-to-dorsal distal tip cell (DTC) migration defects caused by a weak allele of the netrin receptor gene unc-5. The sdn-1(ev697) allele was identified in a genetic screen for enhancers of unc-5 DTC migration defects, and carried a nonsense mutation predicted to truncate the SDN-1 protein prior to the transmembrane domain. The enhancement of unc-5 caused by an sdn-1 mutation was rescued by expression of wild-type sdn-1 in the hypodermis or nervous system rather than the DTCs, indicating a cell non-autonomous function of sdn-1. The enhancement was also partially reversed by mutations in the egl-17/FGF or egl-20/Wnt genes, suggesting that sdn-1 affects UNC-5 function through a mis-regulation of signaling in growth factor pathways. egl-20 reporter constructs exhibited increased and mis-localized EGL-20 distribution in sdn-1 mutants compared to wild-type animals. Finally, using loss of function mutations, we show that egl-17/Fgf and egl-20/Wnt are partially redundant in regulating the migration pattern of the posterior DTC, as double mutants exhibit significant frequencies of defects in migration phases along both the anteroposterior and dorsoventral axes. Together these results suggest that SDN-1 affects UNC-5 function by regulating the proper extracellular distribution of growth factors.

Molecular and clinical characterization of the myopathic form of mitochondrial DNA depletion syndrome caused by mutations in the thymidine kinase (TK2) gene.

PubMed

Chanprasert, Sirisak; Wang, Jing; Weng, Shao-Wen; Enns, Gregory M; Boué, Daniel R; Wong, Brenda L; Mendell, Jerry R; Perry, Deborah A; Sahenk, Zarife; Craigen, William J; Alcala, Francisco J Climent; Pascual, Juan M; Melancon, Serge; Zhang, Victor Wei; Scaglia, Fernando; Wong, Lee-Jun C

2013-01-01

Mitochondrial DNA (mtDNA) depletion syndromes (MDSs) are a clinically and molecularly heterogeneous group of mitochondrial cytopathies characterized by severe mtDNA copy number reduction in affected tissues. Clinically, MDSs are mainly categorized as myopathic, encephalomyopathic, hepatocerebral, or multi-systemic forms. To date, the myopathic form of MDS is mainly caused by mutations in the TK2 gene, which encodes thymidine kinase 2, the first and rate limiting step enzyme in the phosphorylation of pyrimidine nucleosides. We analyzed 9 unrelated families with 11 affected subjects exhibiting the myopathic form of MDS, by sequencing the TK2 gene. Twelve mutations including 4 novel mutations were detected in 9 families. Skeletal muscle specimens were available from 7 out of 11 subjects. Respiratory chain enzymatic activities in skeletal muscle were measured in 6 subjects, and enzymatic activities were reduced in 3 subjects. Quantitative analysis of mtDNA content in skeletal muscle was performed in 5 subjects, and marked mtDNA content reduction was observed in each. In addition, we outline the molecular and clinical characteristics of this syndrome in a total of 52 patients including those previously reported, and a total of 36 TK2 mutations are summarized. Clinically, hypotonia and proximal muscle weakness are the major phenotypes present in all subjects. In summary, our study expands the molecular and clinical spectrum associated with TK2 deficiency. © 2013.

Novel LAMP2 mutations in Chinese patients with Danon disease cause varying degrees of clinical severity.

PubMed

Luo, Su-shan; Xi, Jian-ying; Cai, Shuang; Zhao, Chong-bo; Lu, Jia-hong; Zhu, Wen-hua; Lin, Jie; Qiao, Kai; Wang, Yin; Ye, Zhu-rong

2014-01-01

Danon disease is an Xlinked dominant lysosomal glycogen storage disorder characterized by cardiomyopathy, skeletal myopathy, and mental retardation. This study described two Chinese cases of Danon disease in order to broaden the phenotypic and genetic spectrum. Clinical data were collected and LAMP2 mutations were analyzed. Patient A had fluctuating limb weakness during 6 months follow-up and was diagnosed with drug-induced myopathy due to anti-hepatitis B therapy with lamivudine. However, the first muscle biopsy with large cytoplasmic vacuoles confused the diagnosis and led to the second biopsy that allowed for the final diagnosis. Patient B had severe cardiac disturbances leading to sudden death. Molecularly, patient A harbored a synonymous mutation adjacent to the exon 6-intron 6 junction; mRNA analysis provided evidence that totally abolished the donor site and caused skipping of exon 6. Patient B harbored a frame-shift deletion mutation in exon 3 (c.396delA) leading to a truncated protein. To our knowledge, this is the first report of Danon disease caused by a synonymous exon mutation that affected mRNA splicing, which indicates that a synonymous substitution may not be silent when it is in the exon sequences close to the splice sites. It is also the first description of Danon disease clinically presenting as druginduced myopathy at onset; the pathological changes might be the key point for making a differential diagnosis. *These two authors contributed equally to this work.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor.

PubMed

Lee, Xiaoyun; Azevedo, Mark D; Armstrong, Donald J; Banowetz, Gary M; Reimmann, Cornelia

2013-02-01

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P. aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E. amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P. fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E. amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Nonlinear dynamics analysis of the human balance control subjected to physical and sensory perturbations.

PubMed

Ashtiani, Mohammed N; Mahmood-Reza, Azghani

2017-01-01

Postural control after applying perturbation involves neural and muscular efforts to limit the center of mass (CoM) motion. Linear dynamical approaches may not unveil all complexities of body efforts. This study was aimed at determining two nonlinear dynamics parameters (fractal dimension (FD) and largest Lyapunov exponent (LLE)) in addition to the linear standing metrics of balance in perturbed stance. Sixteen healthy young males were subjected to sudden rotations of the standing platform. The vision and cognition during the standing were also interfered. Motion capturing was used to measure the lower limb joints and the CoM displacements. The CoM path length as a linear parameter was increased by elimination of vision (p<0.01) and adding a cognitive load (p<0.01). The CoM nonlinear metric FD was decreased due to the cognitive loads (p<0.001). The visual interference increased the FD of all joints when the task included the cognitive loads (p<0.01). The slightly positive LLE values showed weakly-chaotic behavior of the whole body. The local joint rotations indicated higher LLEs. Results indicated weakly chaotic response of the whole body. Increase in the task difficulty by adding sensory interference had difference effects on parameters. Linear and nonlinear metrics of the perturbed stance showed that a combination of them may properly represent the body behavior.

Deconvolution imaging of weak reflective pipe defects using guided-wave signals captured by a scanning receiver.

PubMed

Sun, Zeqing; Sun, Anyu; Ju, Bing-Feng

2017-02-01

Guided-wave echoes from weak reflective pipe defects are usually interfered by coherent noise and difficult to interpret. In this paper, a deconvolution imaging method is proposed to reconstruct defect images from synthetically focused guided-wave signals, with enhanced axial resolution. A compact transducer, circumferentially scanning around the pipe, is used to receive guided-wave echoes from discontinuities at a distance. This method achieves a higher circumferential sampling density than arrayed transducers-up to 72 sampling spots per lap for a pipe with a diameter of 180 mm. A noise suppression technique is used to enhance the signal-to-noise ratio. The enhancement in both signal-to-noise ratio and axial resolution of the method is experimentally validated by the detection of two kinds of artificial defects: a pitting defect of 5 mm in diameter and 0.9 mm in maximum depth, and iron pieces attached to the pipe surface. A reconstructed image of the pitting defect is obtained with a 5.87 dB signal-to-noise ratio. It is revealed that a high circumferential sampling density is important for the enhancement of the inspection sensitivity, by comparing the images reconstructed with different down-sampling ratios. A modified full width at half maximum is used as the criterion to evaluate the circumferential extent of the region where iron pieces are attached, which is applicable for defects with inhomogeneous reflection intensity.

Review of the Diagnosis and Treatment of Periodic Paralysis

PubMed Central

Fontaine, Bertrand; Hanna, Michael G.; Johnson, Nicholas E.; Kissel, John T.; Sansone, Valeria A.; Shieh, Perry B.; Tawil, Rabi N.; Trivedi, Jaya; Cannon, Stephen C.; Griggs, Robert C.

2017-01-01

ABSTRACT Periodic paralyses (PPs) are rare neuromuscular disorders caused by mutations in skeletal muscle sodium, calcium, and potassium channel genes. PPs include hypokalemic paralysis, hyperkalemic paralysis, and Andersen‐Tawil syndrome. Common features of PP include autosomal dominant inheritance, onset typically in the first or second decades, episodic attacks of flaccid weakness, which are often triggered by diet or rest after exercise. Diagnosis is based on the characteristic clinic presentation then confirmed by genetic testing. In the absence of an identified genetic mutation, documented low or high potassium levels during attacks or a decrement on long exercise testing support diagnosis. The treatment approach should include both management of acute attacks and prevention of attacks. Treatments include behavioral interventions directed at avoidance of triggers, modification of potassium levels, diuretics, and carbonic anhydrase inhibitors. Muscle Nerve 57: 522–530, 2018 PMID:29125635

Novel ETF dehydrogenase mutations in a patient with mild glutaric aciduria type II and complex II-III deficiency in liver and muscle

PubMed Central

Wolfe, Lynne A.; He, Miao; Vockley, Jerry; Payne, Nicole; Rhead, William; Hoppel, Charles; Spector, Elaine; Gernert, Kim; Gibson, K. Michael

2014-01-01

We describe a 22-year-old male who developed severe hypoglycemia and lethargy during an acute illness at 4 months of age and subsequently grew and developed normally. At age 4 years he developed recurrent vomiting with mild hyperammonemia and dehydration requiring frequent hospitalizations. Glutaric aciduria Type II was suspected based upon biochemical findings and managed with cornstarch, carnitine and riboflavin supplements. He did not experience metabolic crises between ages 4-12 years. He experienced recurrent vomiting, mild hyperammonemia, and generalized weakness associated with acute illnesses and growth spurts. At age 18 years, he developed exercise intolerance and proximal muscle weakness leading to the identification of multiple acyl-CoAdehydrogenase and complex II/III deficiencies in both skeletal muscle and liver. Subsequent molecular characterization of the ETFDH gene revealed novel heterozygous mutations, p.G274X:c.820 G>T (exon 7) and p.P534L: c.1601 C>T (exon 12), the latter within the iron sulfur-cluster and predicted to affect ubiquinone reductase activity of ETFDH and the docking of ETF to ETFDH. Our case supports the concept of a structural interaction between ETFDH and other enzyme partners, and suggests that the conformational change upon ETF binding to ETFDH may play a key role in linking ETFDH to II/III super-complex formation. PMID:21088898

Novel ETF dehydrogenase mutations in a patient with mild glutaric aciduria type II and complex II-III deficiency in liver and muscle.

PubMed

Wolfe, Lynne A; He, Miao; Vockley, Jerry; Payne, Nicole; Rhead, William; Hoppel, Charles; Spector, Elaine; Gernert, Kim; Gibson, K Michael

2010-12-01

We describe a 22-year-old male who developed severe hypoglycemia and lethargy during an acute illness at 4 months of age and subsequently grew and developed normally. At age 4 years he developed recurrent vomiting with mild hyperammonemia and dehydration requiring frequent hospitalizations. Glutaric aciduria Type II was suspected based upon biochemical findings and managed with cornstarch, carnitine and riboflavin supplements. He did not experience metabolic crises between ages 4-12 years. He experienced recurrent vomiting, mild hyperammonemia, and generalized weakness associated with acute illnesses and growth spurts. At age 18 years, he developed exercise intolerance and proximal muscle weakness leading to the identification of multiple acyl-CoA dehydrogenase and complex II/III deficiencies in both skeletal muscle and liver. Subsequent molecular characterization of the ETFDH gene revealed novel heterozygous mutations, p.G274X:c.820 G > T (exon 7) and p.P534L: c.1601 C > T (exon 12), the latter within the iron sulfur-cluster and predicted to affect ubiquinone reductase activity of ETFDH and the docking of ETF to ETFDH. Our case supports the concept of a structural interaction between ETFDH and other enzyme partners, and suggests that the conformational change upon ETF binding to ETFDH may play a key role in linking ETFDH to II/III super-complex formation.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

2016-10-01

The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

Hereditary motor and sensory neuropathy-russe: new autosomal recessive neuropathy in Balkan Gypsies.

PubMed

Thomas, P K; Kalaydjieva, L; Youl, B; Rogers, T; Angelicheva, D; King, R H; Guergueltcheva, V; Colomer, J; Lupu, C; Corches, A; Popa, G; Merlini, L; Shmarov, A; Muddle, J R; Nourallah, M; Tournev, I

2001-10-01

A novel peripheral neuropathy of autosomal recessive inheritance has been identified in Balkan Gypsies and termed hereditary motor and sensory neuropathy-Russe (HMSN-R). We investigated 21 affected individuals from 10 families. Distal lower limb weakness began between the ages of 8 and 16 years, upper limb involvement beginning between 10 and 43 years, with an average of 22 years. This progressive disorder led to severe weakness of the lower limbs, generalized in the oldest subject (aged 57 years), and marked distal upper limb weakness. Prominent distal sensory loss involved all modalities, resulting in neuropathic joint degeneration in two instances. All patients showed foot deformity, and most showed hand deformity. Motor nerve conduction velocity was moderately reduced in the upper limbs but unobtainable in the legs. Sensory nerve action potentials were absent. There was loss of larger myelinated nerve fibers and profuse regenerative activity in the sural nerve. HMSN-R is a new form of autosomal recessive inherited HMSN caused by a single founder mutation in a 1 Mb interval on chromosome 10q.

Muscle Weakness and Fibrosis Due to Cell Autonomous and Non-cell Autonomous Events in Collagen VI Deficient Congenital Muscular Dystrophy.

PubMed

Noguchi, Satoru; Ogawa, Megumu; Malicdan, May Christine; Nonaka, Ikuya; Nishino, Ichizo

2017-02-01

Congenital muscular dystrophies with collagen VI deficiency are inherited muscle disorders with a broad spectrum of clinical presentation and are caused by mutations in one of COL6A1-3 genes. Muscle pathology is characterized by fiber size variation and increased interstitial fibrosis and adipogenesis. In this study, we define critical events that contribute to muscle weakness and fibrosis in a mouse model with collagen VI deficiency. The Col6a1 GT/GT mice develop non-progressive weakness from younger age, accompanied by stunted muscle growth due to reduced IGF-1 signaling activity. In addition, the Col6a1 GT/GT mice have high numbers of interstitial skeletal muscle mesenchymal progenitor cells, which dramatically increase with repeated myofiber necrosis/regeneration. Our results suggest that impaired neonatal muscle growth and the activation of the mesenchymal cells in skeletal muscles contribute to the pathology of collagen VI deficient muscular dystrophy, and more importantly, provide the insights on the therapeutic strategies for collagen VI deficiency. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The limits of weak selection and large population size in evolutionary game theory.

PubMed

Sample, Christine; Allen, Benjamin

2017-11-01

Evolutionary game theory is a mathematical approach to studying how social behaviors evolve. In many recent works, evolutionary competition between strategies is modeled as a stochastic process in a finite population. In this context, two limits are both mathematically convenient and biologically relevant: weak selection and large population size. These limits can be combined in different ways, leading to potentially different results. We consider two orderings: the [Formula: see text] limit, in which weak selection is applied before the large population limit, and the [Formula: see text] limit, in which the order is reversed. Formal mathematical definitions of the [Formula: see text] and [Formula: see text] limits are provided. Applying these definitions to the Moran process of evolutionary game theory, we obtain asymptotic expressions for fixation probability and conditions for success in these limits. We find that the asymptotic expressions for fixation probability, and the conditions for a strategy to be favored over a neutral mutation, are different in the [Formula: see text] and [Formula: see text] limits. However, the ordering of limits does not affect the conditions for one strategy to be favored over another.

Mutations in the collagen XII gene define a new form of extracellular matrix-related myopathy.

PubMed

Hicks, Debbie; Farsani, Golara Torabi; Laval, Steven; Collins, James; Sarkozy, Anna; Martoni, Elena; Shah, Ashoke; Zou, Yaqun; Koch, Manuel; Bönnemann, Carsten G; Roberts, Mark; Lochmüller, Hanns; Bushby, Kate; Straub, Volker

2014-05-01

Bethlem myopathy (BM) [MIM 158810] is a slowly progressive muscle disease characterized by contractures and proximal weakness, which can be caused by mutations in one of the collagen VI genes (COL6A1, COL6A2 and COL6A3). However, there may be additional causal genes to identify as in ∼50% of BM cases no mutations in the COL6 genes are identified. In a cohort of -24 patients with a BM-like phenotype, we first sequenced 12 candidate genes based on their function, including genes for known binding partners of collagen VI, and those enzymes involved in its correct post-translational modification, assembly and secretion. Proceeding to whole-exome sequencing (WES), we identified mutations in the COL12A1 gene, a member of the FACIT collagens (fibril-associated collagens with interrupted triple helices) in five individuals from two families. Both families showed dominant inheritance with a clinical phenotype resembling classical BM. Family 1 had a single-base substitution that led to the replacement of one glycine residue in the triple-helical domain, breaking the Gly-X-Y repeating pattern, and Family 2 had a missense mutation, which created a mutant protein with an unpaired cysteine residue. Abnormality at the protein level was confirmed in both families by the intracellular retention of collagen XII in patient dermal fibroblasts. The mutation in Family 2 leads to the up-regulation of genes associated with the unfolded protein response (UPR) pathway and swollen, dysmorphic rough-ER. We conclude that the spectrum of causative genes in extracellular matrix (ECM)-related myopathies be extended to include COL12A1.

BAG3-related myopathy, polyneuropathy and cardiomyopathy with long QT syndrome.

PubMed

Kostera-Pruszczyk, Anna; Suszek, Małgorzata; Płoski, Rafał; Franaszczyk, Maria; Potulska-Chromik, Anna; Pruszczyk, Piotr; Sadurska, Elżbieta; Karolczak, Justyna; Kamińska, Anna M; Rędowicz, Maria Jolanta

2015-12-01

BAG3 belongs to BAG family of molecular chaperone regulators interacting with HSP70 and anti-apoptotic protein Bcl-2. It is ubiquitously expressed with strong expression in skeletal and cardiac muscle, and is involved in a panoply of cellular processes. Mutations in BAG3 and aberrations in its expression cause fulminant myopathies, presenting with progressive limb and axial muscle weakness, and respiratory insufficiency and neuropathy. Herein, we report a sporadic case of a 15-years old girl with symptoms of myopathy, demyelinating polyneuropathy and asymptomatic long QT syndrome. Genetic testing demonstrated heterozygous mutation Pro209Leu (c.626C > T) in exon 3 of BAG3 gene causing severe myopathy and neuropathy, often associated with restrictive cardiomyopathy. We did not find a mutation in any known LQT syndrome genes. Analysis of muscle biopsy revealed profound disintegration of Z-discs with extensive accumulation of granular debris and large inclusions within fibers. We demonstrated profound alterations in BAG3 distribution as the protein localized to long filamentous structures present across the fibers that were positively stained not only for α-actinin but also for desmin and filamin indicating that those disintegrated Z-disc regions contained also other sarcomeric proteins. The mutation caused a decrease in the content of BAG3 and HSP70, and also of α-actinin desmin, filamin and fast myosin heavy chain, confirming its severe effect on the muscle fiber morphology and thus function. We provide further evidence that BAG3 is associated with Z-disc maintenance, and the Pro209Leu mutation may occur worldwide. We also provide a summary of cases associated with this mutation reported so far.

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

Mutations of Profilin-1 Associated with Amyotrophic Lateral Sclerosis Promote Aggregation Due to Structural Changes of Its Native State.

PubMed

Del Poggetto, Edoardo; Bemporad, Francesco; Tatini, Francesca; Chiti, Fabrizio

2015-11-20

The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as three mutations, namely C71G, M114T, and G118V, have been found in patients with familial forms of the disease and another, E117G, has been proposed to be a moderate risk factor for disease onset. In this work, we have purified the four profilin-1 variants along with the wild-type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular β-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all four variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation, followed by the G118V and M114T substitutions and then the E117G mutation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all four mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.

Is the canonical RAF-MEK-ERK signaling pathway a therapeutic target in SCLC?

PubMed Central

Cristea, Sandra; Sage, Julien

2017-01-01

The activity of the RAF-MEK-ERK signaling pathway is critical for the proliferation of normal and cancerous cells. Oncogenic mutations driving the development of lung adenocarcinoma often activate this signaling pathway. In contrast, pathway activity levels and their biological roles are not well established in small cell lung cancer (SCLC), a fast-growing neuroendocrine lung cancer subtype. Here we discuss the function of the RAF-MEK-ERK kinase pathway and the mechanisms leading to its activation in SCLC cells. In particular, we argue that activation of this pathway may be beneficial to the survival, proliferation and spread of SCLC cells in response to multiple stimuli. We also consider evidence that high levels of RAF-MEK-ERK pathway activity may be detrimental to SCLC tumors, including in part by interfering with their neuroendocrine fate. Based on these observations, we examine when small molecules targeting kinases in the RAF-MEK-ERK pathway may be useful therapeutically in SCLC patients, including in combination with other therapeutic agents. PMID:27133774

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

NASA Technical Reports Server (NTRS)

Wang, Xiang; Khadpe, Jay; Hu, Baocheng; Iliakis, George; Wang, Ya

2003-01-01

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.

siRNA for Influenza Therapy.

PubMed

Barik, Sailen

2010-07-01

Influenza virus is one of the most prevalent and ancient infections in humans. About a fifth of world's population is infected by influenza virus annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised. In the US alone, influenza outbreaks lead to roughly 30,000 deaths each year. Current vaccines and anti-influenza drugs are of limited use due to high mutation rate of the virus and side effects. In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen. Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication. The siRNAs outperform traditional small molecule antivirals in a number of areas, such as ease of design, modest cost, and fast turnaround. Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism, which is reviewed here.

In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.

PubMed

Watanabe, Tsunamasa; Hatakeyama, Hiroto; Matsuda-Yasui, Chiho; Sato, Yusuke; Sudoh, Masayuki; Takagi, Asako; Hirata, Yuichi; Ohtsuki, Takahiro; Arai, Masaaki; Inoue, Kazuaki; Harashima, Hideyoshi; Kohara, Michinori

2014-04-23

The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.

Identification of the ENT1 antagonists dipyridamole and dilazep as amplifiers of oncolytic herpes simplex virus-1 replication.

PubMed

Passer, Brent J; Cheema, Tooba; Zhou, Bingsen; Wakimoto, Hiroaki; Zaupa, Cecile; Razmjoo, Mani; Sarte, Jason; Wu, Shulin; Wu, Chin-lee; Noah, James W; Li, Qianjun; Buolamwini, John K; Yen, Yun; Rabkin, Samuel D; Martuza, Robert L

2010-05-15

Oncolytic herpes simplex virus-1 (oHSV) vectors selectively replicate in tumor cells, where they kill through oncolysis while sparing normal cells. One of the drawbacks of oHSV vectors is their limited replication and spread to neighboring cancer cells. Here, we report the outcome of a high-throughput chemical library screen to identify small-molecule compounds that augment the replication of oHSV G47Delta. Of the 2,640-screened bioactives, 6 compounds were identified and subsequently validated for enhanced G47Delta replication. Two of these compounds, dipyridamole and dilazep, interfered with nucleotide metabolism by potently and directly inhibiting the equilibrative nucleoside transporter-1 (ENT1). Replicative amplification promoted by dipyridamole and dilazep were dependent on HSV mutations in ICP6, the large subunit of ribonucleotide reductase. Our results indicate that ENT1 antagonists augment oHSV replication in tumor cells by increasing cellular ribonucleoside activity. (c)2010 AACR.

A novel program to design siRNAs simultaneously effective to highly variable virus genomes.

PubMed

Lee, Hui Sun; Ahn, Jeonghyun; Jun, Eun Jung; Yang, Sanghwa; Joo, Chul Hyun; Kim, Yoo Kyum; Lee, Heuiran

2009-07-10

A major concern of antiviral therapy using small interfering RNAs (siRNAs) targeting RNA viral genome is high sequence diversity and mutation rate due to genetic instability. To overcome this problem, it is indispensable to design siRNAs targeting highly conserved regions. We thus designed CAPSID (Convenient Application Program for siRNA Design), a novel bioinformatics program to identify siRNAs targeting highly conserved regions within RNA viral genomes. From a set of input RNAs of diverse sequences, CAPSID rapidly searches conserved patterns and suggests highly potent siRNA candidates in a hierarchical manner. To validate the usefulness of this novel program, we investigated the antiviral potency of universal siRNA for various Human enterovirus B (HEB) serotypes. Assessment of antiviral efficacy using Hela cells, clearly demonstrates that HEB-specific siRNAs exhibit protective effects against all HEBs examined. These findings strongly indicate that CAPSID can be applied to select universal antiviral siRNAs against highly divergent viral genomes.

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.

PubMed

Kamerkar, Sushrut; LeBleu, Valerie S; Sugimoto, Hikaru; Yang, Sujuan; Ruivo, Carolina F; Melo, Sonia A; Lee, J Jack; Kalluri, Raghu

2017-06-22

The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic Kras G12D , a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.

BRCA1 interaction of centrosomal protein Nlp is required for successful mitotic progression.

PubMed

Jin, Shunqian; Gao, Hua; Mazzacurati, Lucia; Wang, Yang; Fan, Wenhong; Chen, Qiang; Yu, Wei; Wang, Mingrong; Zhu, Xueliang; Zhang, Chuanmao; Zhan, Qimin

2009-08-21

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability.

BRCA1 Interaction of Centrosomal Protein Nlp Is Required for Successful Mitotic Progression*♦

PubMed Central

Jin, Shunqian; Gao, Hua; Mazzacurati, Lucia; Wang, Yang; Fan, Wenhong; Chen, Qiang; Yu, Wei; Wang, Mingrong; Zhu, Xueliang; Zhang, Chuanmao; Zhan, Qimin

2009-01-01

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability. PMID:19509300

Clinical potential of nintedanib for the second-line treatment of advanced non-small-cell lung cancer: current evidence.

PubMed

Rothschild, Sacha I

2014-01-01

The therapeutic landscape in non-small-cell lung cancer (NSCLC) is changing. The description of molecular alterations leading to NSCLC carcinogenesis and progression (so-called oncogenic driver mutations) and the development of targeted agents interfering with the tumor-promoting intracellular signaling pathways have improved the outcome for many patients with advanced/metastatic NSCLC. However, many patients with stage IV NSCLC do not have one of the targetable predictive biomarkers, and are therefore in need of classical chemotherapy. This especially applies to squamous cell cancer. A platinum-based doublet chemotherapy is the standard of care for patients with stage IV NSCLC. As second-line therapies, docetaxel, pemetrexed, and the EGFR tyrosine-kinase inhibitor erlotinib have demonstrated benefit in Phase III randomized trials. Recently, the addition of the angiokinase inhibitor nintedanib to docetaxel has proven efficacious, and is a new treatment option in the second-line setting. Preclinical and clinical data of nintedanib for the treatment of lung cancer patients are reviewed here.

Clinical potential of nintedanib for the second-line treatment of advanced non-small-cell lung cancer: current evidence

PubMed Central

Rothschild, Sacha I

2014-01-01

The therapeutic landscape in non-small-cell lung cancer (NSCLC) is changing. The description of molecular alterations leading to NSCLC carcinogenesis and progression (so-called oncogenic driver mutations) and the development of targeted agents interfering with the tumor-promoting intracellular signaling pathways have improved the outcome for many patients with advanced/metastatic NSCLC. However, many patients with stage IV NSCLC do not have one of the targetable predictive biomarkers, and are therefore in need of classical chemotherapy. This especially applies to squamous cell cancer. A platinum-based doublet chemotherapy is the standard of care for patients with stage IV NSCLC. As second-line therapies, docetaxel, pemetrexed, and the EGFR tyrosine-kinase inhibitor erlotinib have demonstrated benefit in Phase III randomized trials. Recently, the addition of the angiokinase inhibitor nintedanib to docetaxel has proven efficacious, and is a new treatment option in the second-line setting. Preclinical and clinical data of nintedanib for the treatment of lung cancer patients are reviewed here. PMID:28210142

Silent polymorphisms in the RYR1 gene do not modify the phenotype of the p.4898 I>T pathogenic mutation in central core disease: a case report

PubMed Central

2014-01-01

Background Central core disease is a congenital myopathy, characterized by presence of central core-like areas in muscle fibers. Patients have mild or moderate weakness, hypotonia and motor developmental delay. The disease is caused by mutations in the human ryanodine receptor gene (RYR1), which encodes a calcium-release channel. Since the RYR1 gene is huge, containing 106 exons, mutation screening has been limited to three ‘hot spots’, with particular attention to the C-terminal region. Recent next- generation sequencing methods are now identifying multiple numbers of variants in patients, in which interpretation and phenotype prevision is difficult. Case presentation In a Brazilian Caucasian family, clinical, histopathological and molecular analysis identified a new case of central core disease in a 48-year female. Sanger sequencing of the C-terminal region of the RYR1 gene identified two different missense mutations: c.14256 A > C polymorphism in exon 98 and c.14693 T > C in exon 102, which have already been described as pathogenic. Trans-position of the 2 mutations was confirmed because patient’s daughter, mother and sister carried only the exon 98’s mutation, a synonymous variant that was subsequently found in the frequency of 013–0,05 of alleles. Further next generation sequencing study of the whole RYR1 gene in the patient revealed the presence of additional 5 common silent polymorphisms in homozygosis and 8 polymorphisms in heterozygosis. Conclusions Considering that patient’s relatives showed no pathologic phenotype, and the phenotype presented by the patient is within the range observed in other central core disease patients with the same mutation, it was concluded that the c.14256 A > C polymorphism alone is not responsible for disease, and the associated additional silent polymorphisms are not acting as modifiers of the primary pathogenic mutation in the affected patient. The case described above illustrates the present reality where new methods for wide genome screening are becoming more accessible and able to identify a great variety of mutations and polymorphisms of unknown function in patients and their families. PMID:25084811

Leigh syndrome T8993C mitochondrial DNA mutation: Heteroplasmy and the first clinical presentation in a Vietnamese family.

PubMed

Weerasinghe, Chamara Arachchighe Lahiru; Bui, Bich-Hong Thi; Vu, Thu Thi; Nguyen, Hong-Loan Thi; Phung, Bao-Khanh; Nguyen, Van-Minh; Pham, Van-Anh; Cao, Vu-Hung; Phan, Tuan-Nghia

2018-05-01

Leigh syndrome is a rare inherited, heterogeneous and progressive neurometabolic disorder that is mainly caused by specific mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). The present study reported a case of childhood Leigh syndrome with a point mutation at bp 8,993 in the mitochondrial ATPase6 gene. A 21‑month‑old male child had developed epilepsy, muscular weakness and vomiting, which was accompanied by high fever. Magnetic resonance imaging indicated typical characteristics of Leigh syndrome, including a symmetric abnormal signal in the dorsal medulla oblongata and Sylvian fissure enlargement in association with an abnormal signal in the periventricular white matter and in the putamina and caudate heads. The diagnosis was further supported with genetic tests including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), sequencing, and quantitative PCR. The patient was found to carry a mitochondrial T8993C (m.T8993C) mutation in peripheral blood with 94.00±1.34% heteroplasmy. Eight of his relatives were also subjected to quantification of the m.T8993C mutation. The percentages of heteroplasmy in samples taken from the grandmother, mother, aunt, cousin 1, and cousin 2 were 16.33±1.67, 66.81±0.85, 71.66±3.22, 87.00±1.79, and 91.24±2.50%, respectively. The mutation was not found in samples taken from the father, the husband of the aunt, or the grandfather of the patient. The obtained data showed that the mutation was maternally inherited and accumulated through generations. Even though the heteroplasmy levels of his mother, aunt, cousin 1, and cousin 2 were relatively high (66.81‑91.24%), they remained asymptomatic, indicating that the threshold at which this mutation shows effects is high. To the best of our knowledge, this is the first report of a case of Leigh syndrome in a Vietnamese individual harboring a mtDNA mutation at the 8,993 bp site, and showing a correlation between the heteroplasmy and clinical phenotype. These findings may be useful in helping to improve the clinical diagnosis and treatment of Leigh syndrome.

Clinical Profile of Statin Intolerance in the Phase 3 GAUSS-2 Study.

PubMed

Cho, Leslie; Rocco, Michael; Colquhoun, David; Sullivan, David; Rosenson, Robert S; Dent, Ricardo; Xue, Allen; Scott, Rob; Wasserman, Scott M; Stroes, Erik

2016-06-01

Recent evidence suggests that statin intolerance may be more common than reported in randomized trials. However, the statin-intolerant population is not well characterized. The goal of this report is to characterize the population enrolled in the phase 3 Goal Achievement after Utilizing an anti-PCSK9 antibody in Statin Intolerant Subjects Study (GAUSS-2; NCT 01763905). GAUSS-2 compared evolocumab, a fully human monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9) to ezetimibe in hypercholesterolemic patients who discontinued statin therapy due to statin-associated muscle symptoms (SAMS). GAUSS-2 was a 12-week, double-blind, placebo-controlled, randomized study that enrolled patients with elevated LDL-C who were either not on a statin or able to tolerate only a low-dose due to SAMS. Patients had received ≥2 statins and were unable to tolerate any statin dose or increase in dose above a specified weekly dose due to SAMS. Three hundred seven patients (mean [SD] age, 62 [10] years; 54 % males) were randomized 2:1 (evolocumab:ezetimibe). Mean (SD) LDL-C was 4.99 (1.51) mmol/L. Patients had used ≥2 (100 %), ≥3 (55 %), or ≥4 (21 %) statins. Coronary artery disease was present in 29 % of patients. Statin-intolerant symptoms were myalgia in 80 % of patients, weakness in 39 %, and more serious complications in 20 %. In 98 % of patients, SAMS interfered with normal daily activity; in 52 %, symptoms precluded moderate exertion. Evaluation of the GAUSS-2 trial population of statin-intolerant patients demonstrates that most patients were high risk with severely elevated LDL-C and many had statin-associated muscle symptoms that interfered with their quality of life.

The Bladder Tumor Suppressor Protein TERE1 (UBIAD1)Modulates Cell Cholesterol: Implications for Tumor Progression

PubMed Central

McGarvey, Terry; Wang, Huiyi; Lal, Priti; Puthiyaveettil, Raghunath; Tomaszewski, John; Sepulveda, Jorge; Labelle, Ed; Weiss, Jayne S.; Nickerson, Michael L.; Kruth, Howard S.; Brandt, Wolfgang; Wessjohann, Ludger A.; Malkowicz, S. Bruce

2011-01-01

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression. PMID:21740188

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

PubMed

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

2014-04-14

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

An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.

PubMed

Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J; Backofen, Rolf; Marchfelder, Anita

2015-02-13

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

PubMed

Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

2017-10-06

BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells

PubMed Central

Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

2017-01-01

BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design. PMID:29113311

An APOC3 3′UTR variant associated with plasma triglycerides levels and coronary heart disease by creating a functional miR-4271 binding site

PubMed Central

Hu, Sen-Lin; Cui, Guang-Lin; Huang, Jin; Jiang, Jian-Gang; Wang, Dao-Wen

2016-01-01

Apolipoprotein C-III (APOC3) is a key regulator of plasma triglycerides levels. Increasing evidence has shown that loss-of-function mutations in APOC3 is associated with reduction in plasma triglycerides levels and will confer a benefit in patients at high risk for cardiovascular disease. However, these favorable mutations were extremely distribution discrepant among different ethnics. In this study, the APOC3 gene was resequenced and we identified a common variant which located in the microRNA-binding site in APOC3 and would affect its expression and the risk of coronary heart disease (CHD). The molecular mechanism was explored. We found that the T allele of rs4225 suppressed APOC3 translation by facilitating miR-4271 binding, but not the G allele. Subjects carrying the GG genotype had higher plasma APOC3 levels (p for trend = 0.03) than those with the TT genotype. Furthermore, the T allele was significantly associated with decreased triglyceride levels [Beta (SE): −0.024 (0.020), P = 0.03]. Finally, the case-control study suggested that the TT genotype resulted in a significant reduction in overall CHD risk [OR, 0.89 (95% confidence interval, 0.77–0.98), P = 0.009]. In conclusion, our results provide evidence that the rs4225 in the 3′-UTR of APOC3 might contribute to the risk of CHD by interfering with miR-4271 binding. PMID:27624799

An APOC3 3'UTR variant associated with plasma triglycerides levels and coronary heart disease by creating a functional miR-4271 binding site.

PubMed

Hu, Sen-Lin; Cui, Guang-Lin; Huang, Jin; Jiang, Jian-Gang; Wang, Dao-Wen

2016-09-14

Apolipoprotein C-III (APOC3) is a key regulator of plasma triglycerides levels. Increasing evidence has shown that loss-of-function mutations in APOC3 is associated with reduction in plasma triglycerides levels and will confer a benefit in patients at high risk for cardiovascular disease. However, these favorable mutations were extremely distribution discrepant among different ethnics. In this study, the APOC3 gene was resequenced and we identified a common variant which located in the microRNA-binding site in APOC3 and would affect its expression and the risk of coronary heart disease (CHD). The molecular mechanism was explored. We found that the T allele of rs4225 suppressed APOC3 translation by facilitating miR-4271 binding, but not the G allele. Subjects carrying the GG genotype had higher plasma APOC3 levels (p for trend = 0.03) than those with the TT genotype. Furthermore, the T allele was significantly associated with decreased triglyceride levels [Beta (SE): -0.024 (0.020), P = 0.03]. Finally, the case-control study suggested that the TT genotype resulted in a significant reduction in overall CHD risk [OR, 0.89 (95% confidence interval, 0.77-0.98), P = 0.009]. In conclusion, our results provide evidence that the rs4225 in the 3'-UTR of APOC3 might contribute to the risk of CHD by interfering with miR-4271 binding.

Evaluation of bilirubin concentration in hemolysed samples, is it really impossible? The altitude-curve cartography approach to interfered assays.

PubMed

Brunori, Paola; Masi, Piergiorgio; Faggiani, Luigi; Villani, Luciano; Tronchin, Michele; Galli, Claudio; Laube, Clarissa; Leoni, Antonella; Demi, Maila; La Gioia, Antonio

2011-04-11

Neonatal jaundice might lead to severe clinical consequences. Measurement of bilirubin in samples is interfered by hemolysis. Over a method-depending cut-off value of measured hemolysis, bilirubin value is not accepted and a new sample is required for evaluation although this is not always possible, especially with newborns and cachectic oncological patients. When usage of different methods, less prone to interferences, is not feasible an alternative recovery method for analytical significance of rejected data might help clinicians to take appropriate decisions. We studied the effects of hemolysis over total bilirubin measurement, comparing hemolysis-interfered bilirubin measurement with the non-interfered value. Interference curves were extrapolated over a wide range of bilirubin (0-30 mg/mL) and hemolysis (H Index 0-1100). Interference "altitude" curves were calculated and plotted. A bimodal acceptance table was calculated. Non-interfered bilirubin of given samples was calculated, by linear interpolation between the nearest lower and upper interference curves. Rejection of interference-sensitive data from hemolysed samples for every method should be based not upon the interferent concentration but upon a more complex algorithm based upon the concentration-dependent bimodal interaction between the interfered analyte and the measured interferent. The altitude-curve cartography approach to interfered assays may help laboratories to build up their own method-dependent algorithm and to improve the trueness of their data by choosing a cut-off value different from the one (-10% interference) proposed by manufacturers. When re-sampling or an alternative method is not available the altitude-curve cartography approach might also represent an alternative recovery method for analytical significance of rejected data. Copyright © 2011 Elsevier B.V. All rights reserved.

Negative psychosocial and heavy physical workloads associated with musculoskeletal pain interfering with normal life in older adults: cross-sectional analysis.

PubMed

Lilje, Stina C; Skillgate, Eva; Anderberg, Peter; Berglund, Johan

2015-07-01

Pain is one of the most frequent reasons for seeking health care, and is thus a public health problem. Although there is a progressive increase in pain and impaired physical function with age, few studies are performed on older adults. The aim of this study was to investigate if there are associations between musculoskeletal pain interfering with normal life in older adults and physical and psychosocial workloads through life. The association of heavy physical workload and negative psychosocial workload and musculoskeletal pain interfering with normal life (SF 12) was analyzed by multiple logistic regression. The model was adjusted for eight background covariates: age, gender, growing-up environment, educational level, if living alone or not, obesity, smoking, and leisure physical activity. Negative psychosocial and heavy physical workloads were independently associated with musculoskeletal pain interfering with normal life (adjusted OR: 4.44, 95% CI: 2.84-6.92), and (adjusted OR: 1.88, 95% CI: 1.20-2.93), respectively. The background covariates female gender and higher education were also associated with musculoskeletal pain interfering with normal life, and physical leisure activity was inversely associated. The findings suggest that negative psychosocial and heavy physical workloads are strongly associated with musculoskeletal pain interfering with normal life in older adults. © 2015 the Nordic Societies of Public Health.

Mutations in GMPPB cause congenital myasthenic syndrome and bridge myasthenic disorders with dystroglycanopathies.

PubMed

Belaya, Katsiaryna; Rodríguez Cruz, Pedro M; Liu, Wei Wei; Maxwell, Susan; McGowan, Simon; Farrugia, Maria E; Petty, Richard; Walls, Timothy J; Sedghi, Maryam; Basiri, Keivan; Yue, Wyatt W; Sarkozy, Anna; Bertoli, Marta; Pitt, Matthew; Kennett, Robin; Schaefer, Andrew; Bushby, Kate; Parton, Matt; Lochmüller, Hanns; Palace, Jacqueline; Muntoni, Francesco; Beeson, David

2015-09-01

Congenital myasthenic syndromes are inherited disorders that arise from impaired signal transmission at the neuromuscular junction. Mutations in at least 20 genes are known to lead to the onset of these conditions. Four of these, ALG2, ALG14, DPAGT1 and GFPT1, are involved in glycosylation. Here we identify a fifth glycosylation gene, GMPPB, where mutations cause congenital myasthenic syndrome. First, we identified recessive mutations in seven cases from five kinships defined as congenital myasthenic syndrome using decrement of compound muscle action potentials on repetitive nerve stimulation on electromyography. The mutations were present through the length of the GMPPB, and segregation, in silico analysis, exon trapping, cell transfection followed by western blots and immunostaining were used to determine pathogenicity. GMPPB congenital myasthenic syndrome cases show clinical features characteristic of congenital myasthenic syndrome subtypes that are due to defective glycosylation, with variable weakness of proximal limb muscle groups while facial and eye muscles are largely spared. However, patients with GMPPB congenital myasthenic syndrome had more prominent myopathic features that were detectable on muscle biopsies, electromyography, muscle magnetic resonance imaging, and through elevated serum creatine kinase levels. Mutations in GMPPB have recently been reported to lead to the onset of muscular dystrophy dystroglycanopathy. Analysis of four additional GMPPB-associated muscular dystrophy dystroglycanopathy cases by electromyography found that a defective neuromuscular junction component is not always present. Thus, we find mutations in GMPPB can lead to a wide spectrum of clinical features where deficit in neuromuscular transmission is the major component in a subset of cases. Clinical recognition of GMPPB-associated congenital myasthenic syndrome may be complicated by the presence of myopathic features, but correct diagnosis is important because affected individuals can respond to appropriate treatments. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.

A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy.

PubMed

Tsai, Pei-Chien; Soong, Bing-Wen; Mademan, Inès; Huang, Yen-Hua; Liu, Chia-Rung; Hsiao, Cheng-Tsung; Wu, Hung-Ta; Liu, Tze-Tze; Liu, Yo-Tsen; Tseng, Yen-Ting; Lin, Kon-Ping; Yang, Ueng-Cheng; Chung, Ki Wha; Choi, Byung-Ok; Nicholson, Garth A; Kennerson, Marina L; Chan, Chih-Chiang; De Jonghe, Peter; Cheng, Tzu-Hao; Liao, Yi-Chu; Züchner, Stephan; Baets, Jonathan; Lee, Yi-Chung

2017-05-01

Distal hereditary motor neuropathy is a heterogeneous group of inherited neuropathies characterized by distal limb muscle weakness and atrophy. Although at least 15 genes have been implicated in distal hereditary motor neuropathy, the genetic causes remain elusive in many families. To identify an additional causal gene for distal hereditary motor neuropathy, we performed exome sequencing for two affected individuals and two unaffected members in a Taiwanese family with an autosomal dominant distal hereditary motor neuropathy in which mutations in common distal hereditary motor neuropathy-implicated genes had been excluded. The exome sequencing revealed a heterozygous mutation, c.770A > G (p.His257Arg), in the cytoplasmic tryptophanyl-tRNA synthetase (TrpRS) gene (WARS) that co-segregates with the neuropathy in the family. Further analyses of WARS in an additional 79 Taiwanese pedigrees with inherited neuropathies and 163 index cases from Australian, European, and Korean distal hereditary motor neuropathy families identified the same mutation in another Taiwanese distal hereditary motor neuropathy pedigree with different ancestries and one additional Belgian distal hereditary motor neuropathy family of Caucasian origin. Cell transfection studies demonstrated a dominant-negative effect of the p.His257Arg mutation on aminoacylation activity of TrpRS, which subsequently compromised protein synthesis and reduced cell viability. His257Arg TrpRS also inhibited neurite outgrowth and led to neurite degeneration in the neuronal cell lines and rat motor neurons. Further in vitro analyses showed that the WARS mutation could potentiate the angiostatic activities of TrpRS by enhancing its interaction with vascular endothelial-cadherin. Taken together, these findings establish WARS as a gene whose mutations may cause distal hereditary motor neuropathy and alter canonical and non-canonical functions of TrpRS. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia

PubMed Central

Rochmijati Wuliandari, Juli; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

2015-01-01

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations. PMID:26463408

Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2

PubMed Central

Foley, A. Reghan; Menezes, Manoj P.; Pandraud, Amelie; Gonzalez, Michael A.; Al-Odaib, Ahmad; Abrams, Alexander J.; Sugano, Kumiko; Yonezawa, Atsushi; Manzur, Adnan Y.; Burns, Joshua; Hughes, Imelda; McCullagh, B. Gary; Jungbluth, Heinz; Lim, Ming J.; Lin, Jean-Pierre; Megarbane, Andre; Urtizberea, J. Andoni; Shah, Ayaz H.; Antony, Jayne; Webster, Richard; Broomfield, Alexander; Ng, Joanne; Mathew, Ann A.; O’Byrne, James J.; Forman, Eva; Scoto, Mariacristina; Prasad, Manish; O’Brien, Katherine; Olpin, Simon; Oppenheim, Marcus; Hargreaves, Iain; Land, John M.; Wang, Min X.; Carpenter, Kevin; Horvath, Rita; Straub, Volker; Lek, Monkol; Gold, Wendy; Farrell, Michael O.; Brandner, Sebastian; Phadke, Rahul; Matsubara, Kazuo; McGarvey, Michael L.; Scherer, Steven S.; Baxter, Peter S.; King, Mary D.; Clayton, Peter; Rahman, Shamima; Reilly, Mary M.; Ouvrier, Robert A.; Christodoulou, John; Züchner, Stephan; Muntoni, Francesco

2014-01-01

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms. PMID:24253200

Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2.

PubMed

Foley, A Reghan; Menezes, Manoj P; Pandraud, Amelie; Gonzalez, Michael A; Al-Odaib, Ahmad; Abrams, Alexander J; Sugano, Kumiko; Yonezawa, Atsushi; Manzur, Adnan Y; Burns, Joshua; Hughes, Imelda; McCullagh, B Gary; Jungbluth, Heinz; Lim, Ming J; Lin, Jean-Pierre; Megarbane, Andre; Urtizberea, J Andoni; Shah, Ayaz H; Antony, Jayne; Webster, Richard; Broomfield, Alexander; Ng, Joanne; Mathew, Ann A; O'Byrne, James J; Forman, Eva; Scoto, Mariacristina; Prasad, Manish; O'Brien, Katherine; Olpin, Simon; Oppenheim, Marcus; Hargreaves, Iain; Land, John M; Wang, Min X; Carpenter, Kevin; Horvath, Rita; Straub, Volker; Lek, Monkol; Gold, Wendy; Farrell, Michael O; Brandner, Sebastian; Phadke, Rahul; Matsubara, Kazuo; McGarvey, Michael L; Scherer, Steven S; Baxter, Peter S; King, Mary D; Clayton, Peter; Rahman, Shamima; Reilly, Mary M; Ouvrier, Robert A; Christodoulou, John; Züchner, Stephan; Muntoni, Francesco; Houlden, Henry

2014-01-01

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia.

PubMed

Wuliandari, Juli Rochmijati; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

2015-07-23

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations.

Polymers in Small-Interfering RNA Delivery

PubMed Central

Singha, Kaushik; Namgung, Ran

2011-01-01

This review will cover the current strategies that are being adopted to efficiently deliver small interfering RNA using nonviral vectors, including the use of polymers such as polyethylenimine, poly(lactic-co-glycolic acid), polypeptides, chitosan, cyclodextrin, dendrimers, and polymers-containing different nanoparticles. The article will provide a brief and concise account of underlying principle of these polymeric vectors and their structural and functional modifications which were intended to serve different purposes to affect efficient therapeutic outcome of small-interfering RNA delivery. The modifications of these polymeric vectors will be discussed with reference to stimuli-responsiveness, target specific delivery, and incorporation of nanoconstructs such as carbon nanotubes, gold nanoparticles, and silica nanoparticles. The emergence of small-interfering RNA as the potential therapeutic agent and its mode of action will also be mentioned in a nutshell. PMID:21749290

Late-Onset Glycogen Storage Disease Type II (Pompe's Disease) with a Novel Mutation: A Malaysian Experience.

PubMed

Fu Liong, Hiew; Abdul Wahab, Siti Aishah; Yakob, Yusnita; Lock Hock, Ngu; Thong, Wong Kum; Viswanathan, Shanthi

2014-01-01

Pompe's disease (acid maltase deficiency, glycogen storage disease type II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid α-1,4-glucosidase, resulting in excessive accumulation of glycogen in the lysosomes and cytoplasm of all tissues, most notably in skeletal muscles. We present a case of adult-onset Pompe's disease with progressive proximal muscles weakness over 5 years and respiratory failure on admission, requiring prolonged mechanical ventilation. Electromyography showed evidence of myopathic process with small amplitudes, polyphasic motor unit action potentials, and presence of pseudomyotonic discharges. Muscle biopsy showed glycogen-containing vacuoles in the muscle fibers consistent with glycogen storage disease. Genetic analysis revealed two compound heterozygous mutations at c.444C>G (p.Tyr148∗) in exon 2 and c.2238G>C (p.Trp746Cys) in exon 16, with the former being a novel mutation. This mutation has not been reported before, to our knowledge. The patient was treated with high protein diet during the admission and subsequently showed good clinical response to enzyme replacement therapy with survival now to the eighth year. Conclusion. In patients with late-onset adult Pompe's disease, careful evaluation and early identification of the disease and its treatment with high protein diet and enzyme replacement therapy improve muscle function and have beneficial impact on long term survival.

Renal dysfunction and barttin expression in Bartter syndrome Type IV associated with a G47R mutation in BSND in a family.

PubMed

Park, C W; Lim, J H; Youn, D-Y; Chung, S; Lim, M-H; Kim, Y K; Chang, Y S; Lee, J-H

2011-02-01

Bartter syndrome (BS) Type IV, associated with a G47R mutation in the BSND gene, is known to result in a mild renal phenotype. However, we report here on three brothers with varying degrees of renal dysfunction from mild to end-stage renal disease associated with renal barttin and ClC-K expression. The brothers had histories of polyhydramnios, prematurity, polyuria, deafness, and small body size. Laboratory findings showed hypokalemic metabolic alkalosis, normotensive hyperreninemic hyperaldosteronism, and an increased urinary excretion of sodium, potassium and chloride, consistent with BS Type IV. Microscopic examination of renal tissue showed hyperplasia of cells at the juxtaglomerular apparatus with dilated atrophic tubules and tubulointerstitial fibrosis. A weak barttin signal related to CIC-K expression in the cytoplasm of tubule cells, but not the basement membrane, was noted. A sequence analysis of the BSND gene showed that the affected males were homozygous for a missense G47R mutation in exon 1 of BSND. These findings suggest that the G47R mutation results in a dramatic decrease in barttin expression, which appears to be related to the location of CIC-K being changed from the basement membrane to the cytoplasm in the tubule and might have varying effects on renal function associated with factors other than this gene.

Muscle Weakness, Cardiomyopathy, and L-2-Hydroxyglutaric Aciduria Associated with a Novel Recessive SLC25A4 Mutation.

PubMed

von Renesse, Anja; Morales-Gonzalez, Susanne; Gill, Esther; Salomons, Gajja S; Stenzel, Werner; Schuelke, Markus

2018-04-14

Mutations in SLC25A4 (syn. ANT1, Adenine nucleotide translocase, type 1) are known to cause either autosomal dominant progressive external ophthalmoplegia (adPEO) or recessive mitochondrial myopathy, hypertrophic cardiomyopathy, and lactic acidosis. Whole exome sequencing in a young man with myopathy, subsarcolemmal mitochondrial aggregations, cardiomyopathy, lactic acidosis, and L-2-hydroxyglutaric aciduria (L-2-HGA) revealed a new homozygous mutation in SLC25A4 [c.653A>C, NM_001151], leading to the replacement of a highly conserved glutamine by proline [p.(Q218P); NP_001142] that most likely affects the folding of the ANT1 protein. No pathogenic mutation was found in L2HGDH, which is associated with "classic" L-2-HGA. Furthermore, L-2-HGDH enzymatic activity in the patient fibroblasts was normal. Long-range PCR and Southern blot confirmed absence of mtDNA-deletions in blood and muscle. The disturbed ADP/ATP transport across the inner mitochondrial membrane may lead to an accumulation of different TCA-cycle intermediates such as 2-ketoglutarate (2-KG) in our patient. As L-2-HG is generated from 2-KG we hypothesize that the L-2-HG increase is a secondary effect of 2-KG accumulation. Hence, our report expands the spectrum of laboratory findings in ANT1-related diseases and hints towards a connection with organic acidurias.

Novel mutations in DNAJB6 gene cause a very severe early-onset limb-girdle muscular dystrophy 1D disease.

PubMed

Palmio, Johanna; Jonson, Per Harald; Evilä, Anni; Auranen, Mari; Straub, Volker; Bushby, Kate; Sarkozy, Anna; Kiuru-Enari, Sari; Sandell, Satu; Pihko, Helena; Hackman, Peter; Udd, Bjarne

2015-11-01

DNAJB6 is the causative gene for limb-girdle muscular dystrophy 1D (LGMD1D). Four different coding missense mutations, p.F89I, p.F93I, p.F93L, and p.P96R, have been reported in families from Europe, North America and Asia. The previously known mutations cause mainly adult-onset proximal muscle weakness with moderate progression and without respiratory involvement. A Finnish family and a British patient have been studied extensively due to a severe muscular dystrophy. The patients had childhood-onset LGMD, loss of ambulation in early adulthood and respiratory involvement; one patient died of respiratory failure aged 32. Two novel mutations, c.271T > A (p.F91I) and c.271T > C (p.F91L), in DNAJB6 were identified by whole exome sequencing as a cause of this severe form of LGMD1D. The results were confirmed by Sanger sequencing. The anti-aggregation effect of the mutant DNAJB6 was investigated in a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin as a model for an aggregation-prone protein. Both novel mutant proteins show a significant loss of ability to prevent aggregation. Copyright © 2015 Elsevier B.V. All rights reserved.

Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

PubMed Central

Xiao, Jing; Kim, Leslie S.

2006-01-01

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly. PMID:16687570

Novel Homozygous Missense Mutation in RYR1 Leads to Severe Congenital Ptosis, Ophthalmoplegia, and Scoliosis in the Absence of Myopathy.

PubMed

Dilaver, Nafi; Mazaheri, Neda; Maroofian, Reza; Zeighami, Jawaher; Seifi, Tahere; Zamani, Mina; Sedaghat, Alireza; Shariati, Gholam Reza; Galehdari, Hamid

2017-12-01

Ryanodine receptor 1 ( RYR1 ) is an intracellular calcium receptor primarily expressed in skeletal muscle with a role in excitation contraction. Both dominant and recessive mutations in the RYR1 gene cause a range of RYR1 -related myopathies and/or susceptibility to malignant hyperthermia (MH). Recently, an atypical manifestation of ptosis, variably presenting with ophthalmoplegia, facial paralysis, and scoliosis but without significant muscle weakness, has been reported in 9 cases from 4 families with bialleic variants in RYR1 . Two affected children from a consanguineous family with severe congenital ptosis, ophthalmoplegia, scoliosis, and distinctive long faces but without skeletal myopathy were studied. To identify the cause of the hereditary condition, DNA from the proband was subjected to whole exome sequencing (WES). WES revealed a novel homozygous missense variant in RYR1 (c.14066T>A; p.IIe4689Asn), which segregated within the family. Although the phenotype of the affected siblings in this study was similar to previously described cases, the clinical features were more severely expressed. Our findings contribute to the expansion of phenotypes related to RYR1 dysfunction. Additionally, it supports a new RYR1 -related clinical presentation without musculoskeletal involvement. It is important that individuals with RYR1 mutations are considered susceptible to MH, as 70% of the MH cases are caused by mutations in the RYR1 gene.