scholarly journals Interpreting TMEM67 missense variants of uncertain significance (VUS) in an animal model

2021 ◽  
Author(s):  
Karen I Lange ◽  
Sunayna Best ◽  
Sofia Tsiropoulou ◽  
Ian Berry ◽  
Colin A Johnson ◽  
...  
Keyword(s):  
Animal Model ◽  
Molecular Genetic ◽  
Genetic Diagnosis ◽  
Knock Out ◽  
Variants Of Uncertain Significance ◽  
C Elegans ◽  
In Vivo Animal Model ◽  
Uncertain Significance

Purpose: A molecular genetic diagnosis is essential for accurate counselling and management of patients with ciliopathies. Uncharacterized missense alleles are often classified as variants of uncertain significance (VUS) and are not clinically useful. In this study, we explore the use of a tractable animal model (C. elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a gene frequently mutated as a cause of ciliopathies. Methods: CRISPR/Cas9 gene editing was used to generate homozygous worm strains carrying TMEM67 patient variants. Quantitative phenotypic assays (dye filling, roaming, chemotaxis) assessed cilia structure and function. Results were validated by genetic complementation assays in a human TMEM67 knock-out hTERT-RPE1 cell line. Results: Quantitative assays in C. elegans distinguished between known benign (Asp359Glu, Thr360Ala) and pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of seven missense VUS alleles predicted two benign (Cys173Arg, Thr176Ile) and four pathogenic variants (Cys170Tyr, His782Arg, Gly786Glu, His790Arg). Results from one VUS (Gly979Arg) were inconclusive in worms, but additional in vitro validation suggested it was likely benign. Conclusion: Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows stratification and rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

Thermo-responsive chitosan hydrogel for healing of full-thickness wounds infected with XDR bacteria isolated from burn patients: In vitro and in vivo animal model

2020 ◽  
Vol 164 ◽  
pp. 4475-4486
Author(s):  
Zahra Aliakbar Ahovan ◽  
Sadjad Khosravimelal ◽  
Behnaz Sadat Eftekhari ◽  
Soraya Mehrabi ◽  
Ali Hashemi ◽  
...  
Keyword(s):  
Animal Model ◽  
Burn Patients ◽  
Full Thickness ◽  
Chitosan Hydrogel ◽  
In Vivo Animal Model

In vitro activity and in vivo animal model efficacy of IB-367 alone and in combination with imipenem and colistin against Gram-negative bacteria

Peptides ◽  
2014 ◽  
Vol 55 ◽  
pp. 17-22 ◽  
Author(s):  
Oriana Simonetti ◽  
Oscar Cirioni ◽  
Roberto Ghiselli ◽  
Fiorenza Orlando ◽  
Carmela Silvestri ◽  
...  
Keyword(s):  
Animal Model ◽  
Vitro Activity ◽  
Gram Negative Bacteria ◽  
In Vitro Activity ◽  
Gram Negative ◽  
In Vivo Animal Model

scholarly journals Influence of Exogenous Estrogen Receptor Ligands on Uterine Leiomyoma: Evidence from an in Vitro/in Vivo Animal Model for Uterine Fibroids

2000 ◽  
Vol 108 ◽  
pp. 829 ◽  
Author(s):  
Deborah S. Hunter ◽  
Leslie C. Hodges ◽  
Patricia K. Eagon ◽  
Peter M. Vonier ◽  
Robin Fuchs-Young ◽  
...  
Keyword(s):  
Animal Model ◽  
Estrogen Receptor ◽  
Uterine Leiomyoma ◽  
Uterine Fibroids ◽  
Receptor Ligands ◽  
Exogenous Estrogen ◽  
In Vivo Animal Model ◽  
Estrogen Receptor Ligands

scholarly journals A Gene Replacement Humanization Platform for Rapid Functional Testing of Clinical Variants in Epilepsy-associated STXBP1.

2021 ◽  
Author(s):  
Kathryn McCormick ◽  
Trisha Brock ◽  
Matthew Wood ◽  
Lan Guo ◽  
Kolt McBride ◽  
...  
Keyword(s):  
Motor Function ◽  
Small Animal ◽  
Support Vector ◽  
Variant Interpretation ◽  
Causative Gene ◽  
Coding Sequence ◽  
Variants Of Uncertain Significance ◽  
C Elegans ◽  
Uncertain Significance

Purpose: Functional evidence is a pillar of variant interpretation according to ACMG guidelines. Functional evidence can be obtained in a variety of models and assay systems, including patient-derived tissues and iPSCs, in vitro cellular assays, and in vivo assays. Here we evaluate the reliability and practicality of variant interpretation in the small animal model, C. elegans, through a series of experiments evaluating the function of syntaxin binding protein, STXBP1, a well-known causative gene for Early infantile epileptic encephalopathy 1 (EIEE1). Methods: Using CRISPR, we replaced the coding sequence for unc-18 with the coding sequence for the human ortholog STXBP1. Next, we used CRISPR to introduce precise point mutations in the human STXBP1 coding sequence, reflecting three clinical categories (benign, pathogenic, and variants of uncertain significance (VUS)). We quantified 26 features of the resulting worms movement to train Random Forest (RF) and Support Vector Machines (SVM) machine learning classifiers on known pathogenic and benign variants. We characterized the classifiers, and then used the behavioral data from the VUS-expressing animals to predict the categorization of the VUS. Results: Whereas knock-out worms without unc-18 are severely impaired in motor function, worms expressing STXBP1 in its place have restored motor function. We produced worms with STXBP1 variants previously classified by ACMG criteria, including 25 benign variants, 32 pathogenic, and 24 variants of uncertain significance (VUS). Using either SVM or RF classifiers, we were able to obtain a sensitivity of 0.84-0.97 on known benign and pathogenic strains. By comparing multiple ML classification methods, we were able to classify 9 of the VUS as functionally abnormal, suggesting that these VUS are likely to be pathogenic. Conclusions: We demonstrate that automated analysis of a small animal system is an effective, scalable, and fast way to understand functional consequences of variants in STXBP1, one of the most common causes of genetic epilepsies and neurodevelopmental disorders. Keywords: STXBP1, C. elegans, CRISPR, Unc-18

scholarly journals Comparative assessment of fluorescent proteins forin vivoimaging in an animal model system

2016 ◽  
Author(s):  
Jennifer K Heppert ◽  
Daniel J Dickinson ◽  
Ariel M Pani ◽  
Christopher D Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamicsin vivo. Recent advances in genome editing have enabled precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential ofin vivoimaging experiments, and they facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been madein vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap we quantitatively assessed fluorescent protein propertiesin vivoin an animal model system. We generated transgenicC. elegansstrains expressing green, yellow, or red fluorescent proteins in embryos, and we imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not brightin vivoas predicted based onin vitrodata, but that mNeonGreen is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imagingin vivoinC. elegansembryos, and they suggest good candidate fluorescent proteins to test in other animal model systems.

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