scholarly journals In silico approach to designing rational metagenomic libraries for functional studies

2017 ◽  
Vol 18 (1) ◽  
Author(s):  
Anna Kusnezowa ◽  
Lars I. Leichert
Keyword(s):  
In Silico ◽  
Functional Studies ◽  
Metagenomic Libraries

scholarly journals Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

2018 ◽  
Author(s):  
Jose Velilla ◽  
Michael Mario Marchetti ◽  
Agnes Toth-Petroczy ◽  
Claire Grosgogeat ◽  
Alexis H Bennett ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
In Silico ◽  
Muscular Atrophy ◽  
Structural Modeling ◽  
Recessive Mutation ◽  
Homozygous Mutation ◽  
Functional Studies ◽  
Whole Exome

AbstractObjectiveThe objective of this study is to identify the genetic cause of disease in a congenital form of congenital spinal muscular atrophy and arthrogryposis (CSMAA).MethodsA 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis and diminished lower limb movement. Whole exome sequencing was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identified variant.ResultsWhole exome sequencing revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. Functional studies by western blot and immunohistochemical analysis show that p.S94L reduces TRPV4 protein stability because of increased cytotoxicity and therefore involves a gain-of-function mechanism.ConclusionThis study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of congenital spinal muscular atrophy and arthrogryposis.

scholarly journals Comparison of Predictive In Silico Tools on Missense Variants in GJB2, GJB6, and GJB3 Genes Associated with Autosomal Recessive Deafness 1A (DFNB1A)

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Vera G. Pshennikova ◽  
Nikolay A. Barashkov ◽  
Georgii P. Romanov ◽  
Fedor M. Teryutin ◽  
Aisen V. Solov’ev ◽  
...  
Keyword(s):  
Clinical Significance ◽  
In Silico ◽  
Autosomal Recessive ◽  
Area Under The Curve ◽  
Roc Curves ◽  
Missense Variants ◽  
Functional Studies ◽  
Connexin Genes ◽  
Predictive Software ◽  
And Control

In silico predictive software allows assessing the effect of amino acid substitutions on the structure or function of a protein without conducting functional studies. The accuracy of in silico pathogenicity prediction tools has not been previously assessed for variants associated with autosomal recessive deafness 1A (DFNB1A). Here, we identify in silico tools with the most accurate clinical significance predictions for missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes associated with DFNB1A. To evaluate accuracy of selected in silico tools (SIFT, FATHMM, MutationAssessor, PolyPhen-2, CONDEL, MutationTaster, MutPred, Align GVGD, and PROVEAN), we tested nine missense variants with previously confirmed clinical significance in a large cohort of deaf patients and control groups from the Sakha Republic (Eastern Siberia, Russia): Сх26: p.Val27Ile, p.Met34Thr, p.Val37Ile, p.Leu90Pro, p.Glu114Gly, p.Thr123Asn, and p.Val153Ile; Cx30: p.Glu101Lys; Cx31: p.Ala194Thr. We compared the performance of the in silico tools (accuracy, sensitivity, and specificity) by using the missense variants in GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) genes associated with DFNB1A. The correlation coefficient (r) and coefficient of the area under the Receiver Operating Characteristic (ROC) curve as alternative quality indicators of the tested programs were used. The resulting ROC curves demonstrated that the largest coefficient of the area under the curve was provided by three programs: SIFT (AUC = 0.833, p = 0.046), PROVEAN (AUC = 0.833, p = 0.046), and MutationAssessor (AUC = 0.833, p = 0.002). The most accurate predictions were given by two tested programs: SIFT and PROVEAN (Ac = 89%, Se = 67%, Sp = 100%, r = 0.75, AUC = 0.833). The results of this study may be applicable for analysis of novel missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes.

scholarly journals Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

2019 ◽  
Vol 5 (2) ◽  
pp. e312 ◽  
Author(s):  
Jose Velilla ◽  
Michael Mario Marchetti ◽  
Agnes Toth-Petroczy ◽  
Claire Grosgogeat ◽  
Alexis H. Bennett ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
In Silico ◽  
Muscular Atrophy ◽  
Immunohistochemical Analysis ◽  
Structural Modeling ◽  
Recessive Mutation ◽  
Heterologous Protein Expression ◽  
Homozygous Mutation ◽  
Arthrogryposis Multiplex Congenita ◽  
Functional Studies

ObjectiveTo identify the genetic cause of disease in a form of congenital spinal muscular atrophy and arthrogryposis (CSMAA).MethodsA 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis, and diminished lower limb movement. Whole-exome sequencing (WES) was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identified variant.ResultsWES revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. Functional studies by Western blot and immunohistochemical analysis show that p.S94L increased TRPV4 activity-based cytotoxicity and resultant decreased TRPV4 expression levels, therefore involves a gain-of-function mechanism.ConclusionsThis study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of CSMAA.

scholarly journals In silico and functional studies of the regulation of the glucocerebrosidase gene

2010 ◽  
Vol 99 (3) ◽  
pp. 275-282 ◽  
Author(s):  
Yotam N. Blech-Hermoni ◽  
Shira G. Ziegler ◽  
Kathleen S. Hruska ◽  
Barbara K. Stubblefield ◽  
Mary E. LaMarca ◽  
...  
Keyword(s):  
In Silico ◽  
Functional Studies ◽  
Glucocerebrosidase Gene

A 35kDa antigenic protein from Shigella flexneri: In silico structural and functional studies

2011 ◽  
Vol 415 (2) ◽  
pp. 229-234 ◽  
Author(s):  
Roy Lee Yung-Hung ◽  
Asma Ismail ◽  
Theam Soon Lim ◽  
Yee Siew Choong
Keyword(s):  
In Silico ◽  
Shigella Flexneri ◽  
Antigenic Protein ◽  
Functional Studies

THREE-DIMENSIONAL DEFINITION OF LEAF MORPHOLOGICAL TRAITS OF ARABIDOPSIS IN SILICO PHENOTYPIC ANALYSIS

2005 ◽  
Vol 03 (02) ◽  
pp. 401-414 ◽  
Author(s):  
ELI KAMINUMA ◽  
NAOHIKO HEIDA ◽  
YUKO TSUMOTO ◽  
MIKI NAKAZAWA ◽  
NOBUHARU GOTO ◽  
...  
Keyword(s):  
In Silico ◽  
Morphological Traits ◽  
Three Dimensional ◽  
In Silico Analysis ◽  
Phenotypic Analysis ◽  
3 Dimensional ◽  
Functional Studies ◽  
Phenotypic Alteration ◽  
Definition Of ◽  
Leaf Morphological Traits

The detection of phenotypic alterations of mutants and variants is one of the bottlenecks that hinder systematic gene functional studies of the model plant Arabidopsis. In an earlier study, we have addressed this problem by proposing a novel methodology for phenome analysis based on in silico analysis of polygon models that are acquired by 3-dimensional (3D) measurement and which precisely reconstruct the actual plant shape. However, 3D quantitative descriptions of morphological traits are rare, whereas conventional 2D descriptions have already been studied but may lack the necessary precision. In this report, we focus on six major leaf morphological traits, which are commonly used in the current manual mutant screens, and propose new 3D quantitative definitions that describe these traits. In experiments to extract the traits, we found significant differences between two variants of Arabidopsis with respect to blade roundness and blade epinasty. Remarkably, the detected difference between variants in the blade roundness trait was undetectable when using conventional 2D descriptions. Thus, the result of the experiment indicates that the proposed definitions with 3D description may lead to new discoveries of phenotypic alteration in gene functional studies that would not be possible using conventional 2D descriptions.

scholarly journals Daam2 driven degradation of VHL promotes gliomagenesis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Wenyi Zhu ◽  
Saritha Krishna ◽  
Cristina Garcia ◽  
Chia-Ching John Lin ◽  
Bartley D Mitchell ◽  
...  
Keyword(s):  
Protein Expression ◽  
In Silico ◽  
Inverse Correlation ◽  
Expression Screening ◽  
Functional Studies ◽  
Vhl Protein ◽  
Biochemical Analyses ◽  
Inactivating Mutations ◽  
Potent Tumor

Von Hippel-Landau (VHL) protein is a potent tumor suppressor regulating numerous pathways that drive cancer, but mutations in VHL are restricted to limited subsets of malignancies. Here we identified a novel mechanism for VHL suppression in tumors that do not have inactivating mutations. Using developmental processes to uncover new pathways contributing to tumorigenesis, we found that Daam2 promotes glioma formation. Protein expression screening identified an inverse correlation between Daam2 and VHL expression across a host of cancers, including glioma. These in silico insights guided corroborating functional studies, which revealed that Daam2 promotes tumorigenesis by suppressing VHL expression. Furthermore, biochemical analyses demonstrate that Daam2 associates with VHL and facilitates its ubiquitination and degradation. Together, these studies are the first to define an upstream mechanism regulating VHL suppression in cancer and describe the role of Daam2 in tumorigenesis.

scholarly journals Unmasking of novel mutations within XK gene that could be used as Diagnostic Markers to Predict McLeod syndrome: Using in silico analysis

2019 ◽  
Author(s):  
Mujahed I. Mustafa ◽  
Mohamed A. Hassan
Keyword(s):  
In Silico ◽  
In Silico Analysis ◽  
Structural Effect ◽  
Novel Mutations ◽  
Multisystem Disorder ◽  
Functional Studies ◽  
Central Nervous Systems ◽  
The One ◽  
And Function ◽  
Silico Analysis

AbstractBackgroundMcLeod neuroacanthocytosis syndrome is a rare X-linked recessive multisystem disorder affecting the peripheral and central nervous systems, red blood cells, and internal organs.MethodsWe carried out in silico analysis of structural effect of each SNP using different bioinformatics tools to predict substitution influence on protein structural and functional level.Result2 novel mutations out of 104 nsSNPs that are found to be deleterious effect on the XK structure and function.ConclusionThe present study provided a novel insight into the understanding of McLeod syndrome, SNPs occurring in coding and non-coding regions, may lead to RNA alterations and should be systematically verified. Functional studies can gain from a preliminary multi-step approach, such as the one proposed here; we prioritize SNPs for further genetic mapping studies. This will be a valuable resource for neurologists, hematologists, and clinical geneticists on this rare and debilitating disease.

Abstract 555: Functional Characterization Uncovered Novel Loss-of-function Mutations in ABCA1

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Xin Bi ◽  
James McParland ◽  
Jian Wang ◽  
Adam D McIntyre ◽  
Robert A Hegele ◽  
...  
Keyword(s):  
In Silico ◽  
Cholesterol Efflux ◽  
Functional Characterization ◽  
In Silico Analysis ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Wild Type ◽  
Functional Studies ◽  
Lipid Efflux

Objectives: ABCA1 encodes the membrane protein ATP-binding cassette transporter A1 (ABCA1), a pivotal player in nascent HDL formation via its ability to facilitate cholesterol and phospholipid efflux to apolipoprotein A-I (ApoA-I). ABCA1 variants are frequently found in subjects with primary hypoalphalipoproteinemia, however, their pathogenicity and causal link with the clinical phenotype are not always known. Methods: In silico analysis (Mutation Assessor, PANTHER, PolyPhen-2, PROVEAN, SIFT, and VEST) were performed to predict the functional consequences of ABCA1 missense variants found in our cohort of hypoalphalipoproteinemia. A subset of novel ABCA1 variants were generated in vitro through site-directed mutagenesis and their abilities in mediating lipid efflux to apoA-I were determined using standard methods. Results: A total of 32 mutations in ABCA1 were identified, among which 15 were classified as missense, 9 as nonsense or frameshift, 7 as intronic, and 1 as ”no-protein”. We selected 5 variants that were labeled as pathogenic or possibly pathogenic by in silico analysis to conduct functional studies. Two newly identified mutations in ABCA1, a nonsense mutation (p.E1005X) and a missense mutation (p.S2046R), resulted in complete loss of the canonical lipid efflux function of ABCA1 (2.5% and 1.8% of wild type cholesterol efflux level respectively). These results were concordant with the phenotypic characteristics of the carriers. Three additional mutations (p.G750W and p.R1341T and p.I1085F) resulted in only a partial loss of function (66-75% of wild type cholesterol efflux level). These results were somewhat discordant with the phenotype of the heterozygote carriers (HDL-C levels of 16, 14 and 38 mg/dl respectively), suggesting the presence of additional causal factors. Conclusions: These results support E1005X and S2046R as ABCA1 loss-of-function mutations and highlight the need to conduct functional studies on unknown variants to determine their pathogenicity.

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