scholarly journals Author response: Integrative analysis of large-scale loss-of-function screens identifies robust cancer-associated genetic interactions

2020 ◽  
Author(s):  
Christopher J Lord ◽  
Niall Quinn ◽  
Colm J Ryan
Keyword(s):  
Large Scale ◽  
Integrative Analysis ◽  
Genetic Interactions ◽  
Author Response ◽  
Loss Of Function

scholarly journals Integrative analysis of large-scale loss-of-function screens identifies robust cancer-associated genetic interactions

2019 ◽  
Author(s):  
Christopher J. Lord ◽  
Niall Quinn ◽  
Colm J. Ryan
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Genetic Interactions ◽  
Molecular Heterogeneity ◽  
Loss Of Function ◽  
Driver Genes ◽  
Lethal Effects ◽  
Synthetic Lethal

AbstractGenetic interactions, such as synthetic lethal effects, can now be systematically identified in cancer cell lines using high-throughput genetic perturbation screens. Despite this advance, few genetic interactions have been reproduced across multiple studies and many appear highly context-specific. Understanding which genetic interactions are robust in the face of the molecular heterogeneity observed in tumours and what factors influence this robustness could streamline the identification of therapeutic targets. Here, we develop a computational approach to identify robust genetic interactions that can be reproduced across independent experiments and across non-overlapping cell line panels. We used this approach to evaluate >140,000 potential genetic interactions involving cancer driver genes and identified 1,520 that are significant in at least one study but only 220 that reproduce across multiple studies. Analysis of these interactions demonstrated that: (i) oncogene addiction effects are more robust than oncogene-related synthetic lethal effects; and (ii) robust genetic interactions in cancer are enriched for gene pairs whose protein products physically interact. This suggests that protein-protein interactions can be used not only to understand the mechanistic basis of genetic interaction effects, but also to prioritise robust targets for further development. To explore the utility of this approach, we used a protein-protein interaction network to guide the search for robust synthetic lethal interactions associated with passenger gene alterations and validated two novel robust synthetic lethalities.

scholarly journals Integrative analysis of large-scale loss-of-function screens identifies robust cancer-associated genetic interactions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Christopher J Lord ◽  
Niall Quinn ◽  
Colm J Ryan
Keyword(s):  
Protein Interaction ◽  
Large Scale ◽  
Interaction Network ◽  
Genetic Interactions ◽  
Driver Gene ◽  
Loss Of Function ◽  
Lethal Effects ◽  
Synthetic Lethal ◽  
Protein Protein Interaction ◽  
Context Specific

Genetic interactions, including synthetic lethal effects, can now be systematically identified in cancer cell lines using high-throughput genetic perturbation screens. Despite this advance, few genetic interactions have been reproduced across multiple studies and many appear highly context-specific. Here, by developing a new computational approach, we identified 220 robust driver-gene associated genetic interactions that can be reproduced across independent experiments and across non-overlapping cell line panels. Analysis of these interactions demonstrated that: (i) oncogene addiction effects are more robust than oncogene-related synthetic lethal effects; and (ii) robust genetic interactions are enriched among gene pairs whose protein products physically interact. Exploiting the latter observation, we used a protein–protein interaction network to identify robust synthetic lethal effects associated with passenger gene alterations and validated two new synthetic lethal effects. Our results suggest that protein–protein interaction networks can be used to prioritise therapeutic targets that will be more robust to tumour heterogeneity.

Suppressors of a Cold-Sensitive Mutation in Yeast U4 RNA Define Five Domains in the Splicing Factor Prp8 That Influence Spliceosome Activation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1667-1682 ◽  
Author(s):  
Andreas N Kuhn ◽  
David A Brow
Keyword(s):  
Large Scale ◽  
Splicing Factor ◽  
Cold Sensitivity ◽  
Splicing Factors ◽  
Loss Of Function ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Cold Sensitive ◽  
Spliceosome Activation ◽  
U4 Rna

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

scholarly journals Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot

2021 ◽  
Author(s):  
Doris Škorić-Milosavljević ◽  
Najim Lahrouchi ◽  
Fernanda M. Bosada ◽  
Gregor Dombrowsky ◽  
Simon G. Williams ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Tetralogy Of Fallot ◽  
Large Scale ◽  
Rare Variants ◽  
Growth Factor Receptor ◽  
Vegf Receptor ◽  
Loss Of Function ◽  
Endothelial Growth Factor

Abstract Purpose Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. Methods We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. Results Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). Conclusion Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

BDV-syndrome: An emerging syndrome with profound obesity and neurodevelopmental delay resembling Prader-Willi syndrome

2021 ◽  
Author(s):  
Elisabeth Bosch ◽  
Moritz Hebebrand ◽  
Bernt Popp ◽  
Theresa Penger ◽  
Bettina Behring ◽  
...  
Keyword(s):  
Clinical Features ◽  
Large Scale ◽  
Hypogonadotropic Hypogonadism ◽  
Computational Modelling ◽  
Prader Willi Syndrome ◽  
Loss Of Function ◽  
Human Phenotype ◽  
Neurodevelopmental Delay ◽  
Missense Variants ◽  
Severely Obese

Abstract Context CPE encodes carboxypeptidase E, an enzyme which converts proneuropeptides and propeptide hormones to bioactive forms. It is widely expressed in the endocrine and central nervous system. To date, four individuals from two families with core clinical features including morbid obesity, neurodevelopmental delay and hypogonadotropic hypogonadism, harbouring biallelic loss-of-function CPE variants, were reported. Objective We describe four affected individuals from three unrelated consanguineous families, two siblings of Syrian, one of Egyptian and one of Pakistani descent, all harbouring novel homozygous CPE loss-of-function variants. Methods After excluding Prader-Willi syndrome, exome sequencing was performed in both Syrian siblings. The variants identified in the other two individuals were reported as research variants in a large scale exome study and in ClinVar database. Computational modelling of all possible missense alterations allowed assessing CPE tolerance to missense variants. Results All affected individuals were severely obese with neurodevelopmental delay and other endocrine anomalies. Three individuals from two families shared the same CPE homozygous truncating variant c.361C>T, p.(Arg121*), while the fourth carried the c.994del, p.(Ser333Alafs*22) variant. Comparison of clinical features with previously described cases and standardization according to the Human Phenotype Ontology indicated a recognisable clinical phenotype, which we termed Blakemore-Durmaz-Vasileiou (BDV) syndrome. Computational analysis indicated high conservation of CPE domains and intolerance to missense changes. Conclusions Biallelic truncating CPE variants are associated with BDV syndrome, a clinically recognisable monogenic recessive syndrome with childhood-onset obesity, neurodevelopmental delay, hypogonadotropic hypogonadism and hypothyroidism. BDV syndrome resembles Prader-Willi syndrome. Our findings suggested that missense variants may also be clinically relevant.

Interacting genes required for pharyngeal excitation by motor neuron MC in Caenorhabditis elegans.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1365-1382 ◽  
Author(s):  
D M Raizen ◽  
R Y Lee ◽  
L Avery
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Interactions ◽  
Neuron Type ◽  
Loss Of Function ◽  
Ach Release ◽  
Pharyngeal Muscle ◽  
Recessive Mutations ◽  
Allele Specific ◽  
Pharyngeal Pumping ◽  
Pumping Rates

Abstract We studied the control of pharyngeal excitation in Caenorhabditis elegans. By laser ablating subsets of the pharyngeal nervous system, we found that the MC neuron type is necessary and probably sufficient for rapid pharyngeal pumping. Electropharyngeograms showed that MC transmits excitatory postsynaptic potentials, suggesting that MC acts as a neurogenic pacemaker for pharyngeal pumping. Mutations in genes required for acetylcholine (ACh) release and an antagonist of the nicotinic ACh receptor (nAChR) reduced pumping rates, suggesting that a nAChR is required for MC transmission. To identify genes required for MC neurotransmission, we screened for mutations that cause slow pumping but no other defects. Mutations in two genes, eat-2 and eat-18, eliminated MC neurotransmission. A gain-of-function eat-18 mutation, ad820sd, and a putative loss-of-function eat-18 mutation, ad1110, both reduced the excitation of pharyngeal muscle in response to the nAChR agonists nicotine and carbachol, suggesting that eat-18 is required for the function of a pharyngeal nAChR. Fourteen recessive mutations in eat-2 fell into five complementation classes. We found allele-specific genetic interactions between eat-2 and eat-18 that correlated with complementation classes of eat-2. We propose that eat-18 and eat-2 function in a multisubunit protein complex involved in the function of a pharyngeal nAChR.

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