scholarly journals In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes

2021 ◽  
Author(s):  
Xinbao Ding ◽  
Priti Singh ◽  
Tina N. Tran ◽  
Robert Fragoza ◽  
Haiyuan Yu ◽  
...  
Keyword(s):  
Mouse Models ◽  
High Throughput Sequencing ◽  
Genetic Diagnosis ◽  
Missense Variants ◽  
Protein Protein Interaction ◽  
Prediction Algorithms ◽  
Uncertain Significance ◽  
Human Reproductive ◽  
Fertility Genes

Infertility is a highly heterogeneous condition, with genetic causes estimated to be involved in approximately half of the cases. High-throughput sequencing (HTS) approaches are becoming an increasingly important tool for genetic diagnosis of diseases, including idiopathic infertility. Nevertheless, most rare or minor alleles revealed by HTS are classified as variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of segregating polymorphisms in key fertility genes, we functionally evaluated 8 missense variants in the genes ANKRD31, BRDT, DMC1, EXOI, FKBP6, MSH4 and SEPT12 by generating genome-edited mouse models. Six variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction in the yeast 2 hybrid assay. Even though these genes are known to be essential for normal meiosis or spermiogenesis in mice, none of the tested human variants compromised fertility or gametogenesis in the mouse models. These results should be useful for genetic diagnoses of infertile patients, but they also underscore the need for more effective VUS categorization. To this end, we evaluated the performance of 10 widely used pathogenicity prediction algorithms in classifying missense variants within fertility-related genes from two sources: 1) the ClinVar database, and 2) those functionally tested in mice. We found that all the algorithms performed poorly in terms of predicting phenotypes of mouse-modeled variants. These studies highlight the importance of functional validation of potential infertility-causing genetic variants.

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.

scholarly journals Mouse models of altered gonadotrophin action: insight into male reproductive disorders

Reproduction ◽  
2014 ◽  
Vol 148 (4) ◽  
pp. R63-R70 ◽  
Author(s):  
Kim C Jonas ◽  
Olayiwola O Oduwole ◽  
Hellevi Peltoketo ◽  
Susana B Rulli ◽  
Ilpo T Huhtaniemi
Keyword(s):  
Mouse Models ◽  
Molecular Mechanisms ◽  
Pituitary Hormones ◽  
Loss Of Function ◽  
Reproductive Disorders ◽  
Human Reproductive ◽  
Multiple Loss ◽  
The Relationship

The advent of technologies to genetically manipulate the mouse genome has revolutionised research approaches, providing a unique platform to study the causality of reproductive disorders in vivo. With the relative ease of generating genetically modified (GM) mouse models, the last two decades have yielded multiple loss-of-function and gain-of-function mutation mouse models to explore the role of gonadotrophins and their receptors in reproductive pathologies. This work has provided key insights into the molecular mechanisms underlying reproductive disorders with altered gonadotrophin action, revealing the fundamental roles of these pituitary hormones and their receptors in the hypothalamic–pituitary–gonadal axis. This review will describe GM mouse models of gonadotrophins and their receptors with enhanced or diminished actions, specifically focusing on the male. We will discuss the mechanistic insights gained from these models into male reproductive disorders, and the relationship and understanding provided into male human reproductive disorders originating from altered gonadotrophin action.

scholarly journals High-Throughput Sequencing to Identify Mutations Associated with Retinal Dystrophies

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1269
Author(s):  
Fei Song ◽  
Marta Owczarek-Lipska ◽  
Tim Ahmels ◽  
Marius Book ◽  
Sabine Aisenbrey ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Genetic Diagnosis ◽  
Clinical Manifestations ◽  
Sequence Variants ◽  
Pathogenic Variants ◽  
Retinal Dystrophies ◽  
Whole Exome ◽  
Uncertain Significance ◽  
Disease Associated Genes ◽  
Intronic Variants

Retinal dystrophies (RD) are clinically and genetically heterogenous disorders showing mutations in over 270 disease-associated genes. Several millions of people worldwide are affected with different types of RD. Studying the relevance of disease-associated sequence alterations will assist in understanding disorders and may lead to the development of therapeutic approaches. Here, we established a whole exome sequencing (WES) pipeline to rapidly identify disease-associated mutations in patients. Sanger sequencing was applied to identify deep-intronic variants and to verify the co-segregation of WES results within families. We analyzed 26 unrelated patients with different syndromic and non-syndromic clinical manifestations of RD. All patients underwent ophthalmic examinations. We identified nine novel disease-associated sequence variants among 37 variants identified in total. The sequence variants located to 17 different genes. Interestingly, two cases presenting with Stargardt disease carried deep-intronic variants in ABCA4. We have classified 21 variants as pathogenic variants, 4 as benign/likely benign variants, and 12 as variants of uncertain significance. This study highlights the importance of WES-based mutation analyses in RD patients supporting clinical decisions, broadly based genetic diagnosis and support genetic counselling. It is essential for any genetic therapy to expand the mutation spectrum, understand the genes’ function, and correlate phenotypes with genotypes.

scholarly journals In vivo interactome profiling by enzyme‐catalyzed proximity labeling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yangfan Xu ◽  
Xianqun Fan ◽  
Yang Hu
Keyword(s):  
State Of The Art ◽  
Catalytic Efficiency ◽  
Biological Processes ◽  
Protein Protein Interaction ◽  
Current State ◽  
Protein Interactome ◽  
Potential Applications ◽  
Protein Protein Interaction Networks ◽  
Temporal And Spatial

AbstractEnzyme-catalyzed proximity labeling (PL) combined with mass spectrometry (MS) has emerged as a revolutionary approach to reveal the protein-protein interaction networks, dissect complex biological processes, and characterize the subcellular proteome in a more physiological setting than before. The enzymatic tags are being upgraded to improve temporal and spatial resolution and obtain faster catalytic dynamics and higher catalytic efficiency. In vivo application of PL integrated with other state of the art techniques has recently been adapted in live animals and plants, allowing questions to be addressed that were previously inaccessible. It is timely to summarize the current state of PL-dependent interactome studies and their potential applications. We will focus on in vivo uses of newer versions of PL and highlight critical considerations for successful in vivo PL experiments that will provide novel insights into the protein interactome in the context of human diseases.

627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A663-A663
Author(s):  
Keegan Cooke ◽  
Juan Estrada ◽  
Jinghui Zhan ◽  
Jonathan Werner ◽  
Fei Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mouse Models ◽  
T Cell Activation ◽  
Phase 1 ◽  
Phase 1 Study ◽  
Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

scholarly journals The food additive EDTA aggravates colitis and colon carcinogenesis in mouse models

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rayko Evstatiev ◽  
Adam Cervenka ◽  
Tina Austerlitz ◽  
Gunther Deim ◽  
Maximilian Baumgartner ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Mouse Models ◽  
Bowel Disease ◽  
Intestinal Inflammation ◽  
Food Additives ◽  
Colorectal Carcinogenesis ◽  
Testing Procedures ◽  
Inflammatory Models ◽  
Inflammatory Bowel

AbstractInflammatory bowel disease is a group of conditions with rising incidence caused by genetic and environmental factors including diet. The chelator ethylenediaminetetraacetate (EDTA) is widely used by the food and pharmaceutical industry among numerous other applications, leading to a considerable environmental exposure. Numerous safety studies in healthy animals have revealed no relevant toxicity by EDTA. Here we show that, in the presence of intestinal inflammation, EDTA is surprisingly capable of massively exacerbating inflammation and even inducing colorectal carcinogenesis at doses that are presumed to be safe. This toxicity is evident in two biologically different mouse models of inflammatory bowel disease, the AOM/DSS and the IL10−/− model. The mechanism of this effect may be attributed to disruption of intercellular contacts as demonstrated by in vivo confocal endomicroscopy, electron microscopy and cell culture studies. Our findings add EDTA to the list of food additives that might be detrimental in the presence of intestinal inflammation, but the toxicity of which may have been missed by regulatory safety testing procedures that utilize only healthy models. We conclude that the current use of EDTA especially in food and pharmaceuticals should be reconsidered. Moreover, we suggest that intestinal inflammatory models should be implemented in the testing of food additives to account for the exposure of this primary organ to environmental and dietary stress.

1,25(OH) 2 D 3 attenuates sleep disturbance in mouse models of Lewis lung cancer, in silico and in vivo

2021 ◽  
Author(s):  
Min Ai ◽  
Shuang‐shuang Li ◽  
Hong Chen ◽  
Xi‐ting Wang ◽  
Jiang‐nan Sun ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Sleep Disturbance ◽  
Mouse Models ◽  
In Silico ◽  
Lewis Lung ◽  
Lewis Lung Cancer

scholarly journals Perspectives on Non-BLT Humanized Mouse Models for Studying HIV Pathogenesis and Therapy

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 776
Author(s):  
Kazutaka Terahara ◽  
Ryutaro Iwabuchi ◽  
Yasuko Tsunetsugu-Yokota
Keyword(s):  
Mouse Models ◽  
Humanized Mice ◽  
Lymphoid Tissues ◽  
Humanized Mouse ◽  
Hiv Pathogenesis ◽  
Hematopoietic Stem ◽  
Critical Issues ◽  
In Vivo Animal Models ◽  
Humanized Mouse Models

A variety of humanized mice, which are reconstituted only with human hematopoietic stem cells (HSC) or with fetal thymus and HSCs, have been developed and widely utilized as in vivo animal models of HIV-1 infection. The models represent some aspects of HIV-mediated pathogenesis in humans and are useful for the evaluation of therapeutic regimens. However, there are several limitations in these models, including their incomplete immune responses and poor distribution of human cells to the secondary lymphoid tissues. These limitations are common in many humanized mouse models and are critical issues that need to be addressed. As distinct defects exist in each model, we need to be cautious about the experimental design and interpretation of the outcomes obtained using humanized mice. Considering this point, we mainly characterize the current conventional humanized mouse reconstituted only with HSCs and describe past achievements in this area, as well as the potential contributions of the humanized mouse models for the study of HIV pathogenesis and therapy. We also discuss the use of various technologies to solve the current problems. Humanized mice will contribute not only to the pre-clinical evaluation of anti-HIV regimens, but also to a deeper understanding of basic aspects of HIV biology.

Sign in / Sign up

Export Citation Format

Share Document