scholarly journals Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations

Neurology ◽  
2017 ◽  
Vol 89 (8) ◽  
pp. 762-770 ◽  
Author(s):  
Huijie Feng ◽  
Benita Sjögren ◽  
Behirda Karaj ◽  
Vincent Shaw ◽  
Aysegul Gezer ◽  
...  
Keyword(s):  
Movement Disorder ◽  
Movement Disorders ◽  
Adrenergic Receptor ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Site Directed Mutagenesis ◽  
De Novo Mutations ◽  
Gain Of Function ◽  
Mammalian Expression

Objective:To define molecular mechanisms underlying the clinical spectrum of epilepsy and movement disorder in individuals with de novo mutations in the GNAO1 gene.Methods:We identified all GNAO1 mutations reported in individuals with epilepsy (early infantile epileptiform encephalopathy 17) or movement disorders through April 2016; 15 de novo mutant alleles from 25 individuals were introduced into the Gαo subunit by site-directed mutagenesis in a mammalian expression plasmid. We assessed protein expression and function in vitro in HEK-293T cells by Western blot and determined functional Gαo-dependent cyclic adenosine monophosphate (cAMP) inhibition with a coexpressed α2A adrenergic receptor.Results:Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and loss of function (LOF; <90% maximal inhibition). Six other mutations show variable levels of expression but exhibit normal or even gain-of-function (GOF) behavior, as demonstrated by significantly lower EC50 values for α2A adrenergic receptor–mediated inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic encephalopathy while GOF mutants (such as G42R, G203R, and E246K) or normally functioning mutants (R209) were found in patients with movement disorders with or without seizures.Conclusions:Both LOF and GOF mutations in Gαo (encoded by GNAO1) are associated with neurologic pathophysiology. There appears to be a strong predictive correlation between the in vitro biochemical phenotype and the clinical pattern of epilepsy vs movement disorder.

scholarly journals A gain-of-function mutation in the GRIK2 gene causes neurodevelopmental deficits

2017 ◽  
Vol 3 (1) ◽  
pp. e129 ◽  
Author(s):  
Yomayra F. Guzmán ◽  
Keri Ramsey ◽  
Jacob R. Stolz ◽  
David W. Craig ◽  
Mathew J. Huentelman ◽  
...  
Keyword(s):  
De Novo ◽  
Receptor Gene ◽  
Expression System ◽  
Functional Characterization ◽  
Cell Voltage ◽  
De Novo Mutations ◽  
Gain Of Function ◽  
Whole Exome ◽  
Neurodevelopmental Abnormalities

Objective:To identify inherited or de novo mutations associated with a suite of neurodevelopmental abnormalities in a 10-year-old patient displaying ataxia, motor and speech delay, and intellectual disability.Methods:We performed whole-exome sequencing of the proband and her parents. A pathogenic gene variant was identified as damaging based on sequence conservation, gene function, and association with disorders having similar phenotypic profiles. Functional characterization of the mutated protein was performed in vitro using a heterologous expression system.Results:A single de novo point mutation in the GRIK2 gene was identified as causative for the neurologic symptoms of the proband. The mutation is predicted to change a codon for alanine to that of a threonine at position 657 (A657T) in the GluK2 kainate receptor (KAR) subunit, a member of the ionotropic glutamate receptor gene family. Whole-cell voltage-clamp recordings revealed that KARs incorporating the GluK2(A657T) subunits show profoundly altered channel gating and are constitutively active in nominally glutamate-free extracellular media.Conclusions:In this study, we associate a de novo gain-of-function mutation in the GRIK2 gene with deficits in motor and higher order cognitive function. These results suggest that disruption of physiologic KAR function precludes appropriate development of the nervous system.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

Targeting MDM2-dependent serine metabolism as a therapeutic strategy for liposarcoma

2020 ◽  
Vol 12 (547) ◽  
pp. eaay2163
Author(s):  
Madi Y. Cissé ◽  
Samuel Pyrdziak ◽  
Nelly Firmin ◽  
Laurie Gayte ◽  
Maud Heuillet ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Negative Regulator ◽  
Nucleotide Synthesis ◽  
Metabolic Functions ◽  
Xenograft Models ◽  
Well Differentiated ◽  
Serine Metabolism

Well-differentiated and dedifferentiated liposarcomas (LPSs) are characterized by a systematic amplification of the MDM2 oncogene, which encodes a key negative regulator of the p53 pathway. The molecular mechanisms underlying MDM2 overexpression while sparing wild-type p53 in LPS remain poorly understood. Here, we show that the p53-independent metabolic functions of chromatin-bound MDM2 are exacerbated in LPS and mediate an addiction to serine metabolism that sustains nucleotide synthesis and tumor growth. Treatment of LPS cells with Nutlin-3A, a pharmacological inhibitor of the MDM2-p53 interaction, stabilized p53 but unexpectedly enhanced MDM2-mediated control of serine metabolism by increasing its recruitment to chromatin, likely explaining the poor clinical efficacy of this class of MDM2 inhibitors. In contrast, genetic or pharmacological inhibition of chromatin-bound MDM2 by SP141, a distinct MDM2 inhibitor triggering its degradation, or interfering with de novo serine synthesis, impaired LPS growth both in vitro and in clinically relevant patient-derived xenograft models. Our data indicate that targeting MDM2 functions in serine metabolism represents a potential therapeutic strategy for LPS.

scholarly journals A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 46-48
Author(s):  
M Mehta ◽  
L Wang ◽  
C Guo ◽  
N Warner ◽  
Q Li ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Receptor Tyrosine Kinase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Intestinal Inflammation ◽  
De Novo ◽  
Single Gene ◽  
Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

scholarly journals Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

2019 ◽  
Vol 295 (2) ◽  
pp. 597-609 ◽  
Author(s):  
Taeho Kim ◽  
Peter J. Stogios ◽  
Anna N. Khusnutdinova ◽  
Kayla Nemr ◽  
Tatiana Skarina ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Thermotoga Maritima ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Bacillus Halodurans ◽  
Active Site Residues ◽  
Carbon Carbon Bond ◽  
Condensation Activity

Carbon–carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli. The crystal structures of BH1352 and TM1559 at 1.40–2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

Recurrent De Novo Mutations of EPOR Gene in Primary Congenital Polycythemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1297-1297
Author(s):  
Mariluz P. Mojica-Henshaw ◽  
Caroline Laverdiere ◽  
Jaroslav F. Prchal ◽  
Josef T. Prchal
Keyword(s):  
De Novo ◽  
Stop Codon ◽  
Cell Mass ◽  
Hemoglobin Concentration ◽  
Erythropoietin Receptor ◽  
De Novo Mutations ◽  
Erythroid Progenitors ◽  
Increased Risk ◽  
Genetic Mosaicism

Abstract Primary familial and congenital polycythemia (PFCP) is a rare inherited disorder presenting with elevated red blood cell mass, elevated hemoglobin concentration and low levels of erythropoietin. Ten mutations in the erythropoietin receptor (EPOR) gene to date have been associated with PFCP. All of these mutations result in deletion of 59 to 82 amino acids from the carboxy terminal of EpoR which has been shown to contain a negative regulatory domain. Here, we describe a 2-year old boy of French-Canadian descent presenting with polycythemia and splenomegaly. Sequencing of the EPOR gene showed the proband to be heterozygous for a G to A transition in nucleotide 6002 (G6002A). The mutation generates a stop codon instead of tryptophan at amino acid 439, leading to a truncated EpoR. The association of the G6002A mutation in the EPOR gene with PFCP has been previously described in a large Finnish family (dela Chapelle et al., Proc Natl Acad Sci USA1993; 90: 4495) and in a 16-year old boy of English descent (Percy et al., Br J Hematol1998; 100:407). The G6002A mutation in both cases was considered to have arisen independently based on differences in a microsatellite polymorphism in the 5′UT of EPOR and the absence of the mutation in the immediate family of the English boy. We studied our proband’s parents for the G6002A EPOR mutation and did not find it. Their parentage was confirmed using 24 different microsatellite markers. This indicates that the G6002A mutation in the proband arose de novo. Since the mutation arose de novo, in vitro methycellulose cultures of erythroid progenitors isolated from peripheral blood of the proband were grown in the presence of increasing concentrations of Epo to rule out genetic mosaicism. The erythroid progenitors showed hypersensitivity to Epo as is characteristic of PFCP. However, we did not find evidence supportive of genetic mosaicism as all 70 BFU-E colonies analyzed were heterozygous for the G6002A mutation. Previously, two other polycythemia-associated EPOR mutations, 5974insG (Sokol et al., Blood1995; 86:15) and 5959G>T (Kralovics et al., Am J Hematol2001; 68:115) were shown to have arisen de novo. This case is thus the fourth instance out of 13 reported cases of polycythemia-associated EPOR mutations that has arisen de novo. Because of the rarity of polycythemia-associated EPOR mutations, their frequent de novo occurrence suggests that these mutations do not have a selective advantage but are detrimental. Their possible association with increased risk of thromboembolic and atherosclerotic disease due to chronically augmented Epo signaling is being explored by ongoing clinical studies.

scholarly journals SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

2008 ◽  
Vol 93 (5) ◽  
pp. 1865-1873 ◽  
Author(s):  
Daniel Kelberman ◽  
Sandra C. P. de Castro ◽  
Shuwen Huang ◽  
John A. Crolla ◽  
Rodger Palmer ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Anterior Pituitary ◽  
De Novo ◽  
Hypogonadotropic Hypogonadism ◽  
Expression Patterns ◽  
Critical Role ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Direct Role

Abstract Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye. Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

scholarly journals Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing

2015 ◽  
Vol 25 (3) ◽  
pp. 426-434 ◽  
Author(s):  
Brock A. Peters ◽  
Bahram G. Kermani ◽  
Oleg Alferov ◽  
Misha R. Agarwal ◽  
Mark A. McElwain ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
De Novo ◽  
Whole Genome ◽  
De Novo Mutations ◽  
Single Base

scholarly journals Demonstration of reduced in vivo surface expression of activating mutant thyrotrophin receptors in thyroid sections

2002 ◽  
pp. 163-171 ◽  
Author(s):  
M Sequeira ◽  
B Jasani ◽  
D Fuhrer ◽  
M Wheeler ◽  
M Ludgate
Keyword(s):  
Molecular Mechanisms ◽  
Thyroid Tissue ◽  
Surface Expression ◽  
Germline Mutations ◽  
Antigen Retrieval ◽  
Graves Disease ◽  
Paraffin Sections ◽  
Gain Of Function

OBJECTIVE: Thyroid function and growth are controlled by TSH. Hyperthyroidism can be due to Graves' Disease (GD), in which thyroid-stimulating antibodies mimic TSH, or gain-of-function mutations in the TSH receptor (TSHR). These activating mutations have poor surface expression when assessed in non-thyroidal cells in vitro but nothing is known of their in vivo behaviour. Several TSHR antibodies have been produced but none has been applied to thyroid paraffin sections. This study aimed to develop a technique suitable for use on paraffin sections and apply it to investigate TSHR expression in thyroids harbouring activating TSHR germline mutations compared with normal and GD thyroids. DESIGN AND METHODS: Immunocytochemistry coupled with antigen retrieval, using a spectrum of antibodies to the TSHR, was applied to paraffin sections of GD thyroid tissue. Subsequently, TSHR immunoreactivity was examined in three normal thyroids, three patients with GD and three patients with familial hyperthyroidism, due to different gain-of-function TSHR germline mutations, using the optimised protocol. RESULTS: Two antibodies, A10 and T3-495, to the extracellular domain (ECD) and membrane spanning region (MSR) of the TSHR respectively, produced specific basolateral staining of thyroid follicular cells. In normal and GD thyroids, basolateral staining with T3-495 was generally more intense than with A10, suggesting a possible surfeit of MSR over ECD. Graves' Disease thyroids have more abundant TSHR than normal glands. In contrast, thyroids harbouring gain-of-function mutations have the lowest expression in vivo, mirroring in vitro findings. CONCLUSIONS: The development of an immunocytochemical method applicable to paraffin sections has demonstrated that different molecular mechanisms causing hyperthyroidism result in the lowest (mutation) and highest (autoimmunity) levels of receptor at the thyrocyte surface.

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