scholarly journals A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function

2018 ◽  
Vol 103 (4) ◽  
pp. 498-508 ◽  
Author(s):  
Lea M. Starita ◽  
Muhtadi M. Islam ◽  
Tapahsama Banerjee ◽  
Aleksandra I. Adamovich ◽  
Justin Gullingsrud ◽  
...  
Keyword(s):  
Dna Repair ◽  
Protein Function ◽  
The Impact ◽  
Missense Substitution

scholarly journals A multiplexed homology-directed DNA repair assay reveals the impact of ~1,700 BRCA1 variants on protein function

2018 ◽  
Author(s):  
Lea M. Starita ◽  
Muhtadi M. Islam ◽  
Tapahsama Banerjee ◽  
Aleksandra I. Adamovich ◽  
Justin Gullingsrud ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genetic Testing ◽  
Protein Function ◽  
Missense Variant ◽  
Loss Of Function ◽  
Dna Breaks ◽  
Brca1 Protein ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
The Impact

AbstractLoss-of-function mutations in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for mutations in the BRCA1 gene frequently reveals a missense variant for which the impact on the molecular function of the BRCA1 protein is unknown. Functional BRCA1 is required for homology directed repair (HDR) of double-strand DNA breaks, a key activity for maintaining genome integrity and tumor suppression. Here we describe a multiplex HDR reporter assay to simultaneously measure the effect of hundreds of variants of BRCA1 on its role in DNA repair. Using this assay, we measured the effects of ~1,700 amino acid substitutions in the first 302 residues of BRCA1. Benchmarking these results against variants with known effects, we demonstrate accurate discrimination of loss-of-function versus benign variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing.

Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death

2002 ◽  
Vol 283 (1) ◽  
pp. L205-L210 ◽  
Author(s):  
Allison W. Dobson ◽  
Valentina Grishko ◽  
Susan P. LeDoux ◽  
Mark R. Kelley ◽  
Glenn L. Wilson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Dna Repair ◽  
Trypan Blue Exclusion ◽  
Mt Dna ◽  
Repair Capacity ◽  
Mtdna Damage ◽  
Pulmonary Arterial ◽  
Selective Enhancement ◽  
The Impact

In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the rate of mitochondrial (mt) DNA repair is predictive of the severity of xanthine oxidase (XO)-induced mtDNA damage and the sensitivity to XO-mediated cell death. To examine the importance of mtDNA damage and repair more directly, we determined the impact of mitochondrial overexpression of the DNA repair enzyme, Ogg1, on XO-induced mtDNA damage and cell death in PAECs. PAECs were transiently transfected with an Ogg1-mitochondrial targeting sequence construct. Mitochondria-selective overexpression of the transgene product was confirmed microscopically by the observation that immunoreactive Ogg1 colocalized with a mitochondria-specific tracer and, with an oligonucleotide cleavage assay, by a selective enhancement of mitochondrial Ogg1 activity. Overexpression of Ogg1 protected against both XO-induced mtDNA damage, determined by quantitative Southern analysis, and cell death as assessed by trypan blue exclusion and MTS assays. These findings show that mtDNA damage is a direct cause of cell death in XO-treated PAECs.

scholarly journals Genotype-Phenotype correlation in Dravet Syndrome with SCN1A mutation increase efficiency of molecular diagnosis

2012 ◽  
Vol 18 (2) ◽  
pp. 60-62
Author(s):  
MC Gonsales ◽  
P Preto ◽  
MA Montenegro ◽  
MM Guerreiro ◽  
I Lopes-Cendes
Keyword(s):  
Protein Function ◽  
Copy Number ◽  
Mutation Screening ◽  
Copy Number Variants ◽  
Febrile Seizures ◽  
Copy Number Variations ◽  
Dravet Syndrome ◽  
Missense Mutations ◽  
The Impact ◽  
Doose Syndrome

OBJECTIVES: The purpose of this study was to advance the knowledge on the clinical use of SCN1A testing for severe epilepsies within the spectrum of generalized epilepsy with febrile seizures plus by performing genetic screening in patients with Dravet and Doose syndromes and establishing genotype-phenotype correlations. METHODS: Mutation screening in SCN1A was performed in 15 patients with Dravet syndrome and 13 with Doose syndrome. Eight prediction algorithms were used to analyze the impact of the mutations in putative protein function. Furthermore, all SCN1A mutations previously published were compiled and analyzed. In addition, Multiplex Ligation-Dependent Probe Amplification (MLPA) technique was used to detect possible copy number variations within SCN1A. RESULTS: Twelve mutations were identified in patients with Dravet syndrome, while patients with Doose syndrome showed no mutations. Our results show that the most common type of mutation found is missense, and that they are mostly located in the pore region and the N- and C-terminal of the protein. No copy number variants in SCN1A were identified in our cohort. CONCLUSIONS: SCN1A testing is clinically useful for patients with Dravet syndrome, but not for those with Doose syndrome, since both syndromes do not seem to share the same genetic basis. Our results indicate that indeed missense mutations can cause severe phenotypes depending on its location and the type of amino-acid substitution. Moreover, our strategy for predicting deleterious effect of mutations using multiple computation algorithms was efficient for most of the mutations identified.

scholarly journals Opportunities and Challenges for Interpreting Rare Variation in Clinically Important Genes

2020 ◽  
Author(s):  
Gregory McInnes ◽  
Andrew G. Sharo ◽  
Megan L. Koleske ◽  
Julie E. H. Brown ◽  
Matthew Norstad ◽  
...  
Keyword(s):  
Healthcare System ◽  
Protein Function ◽  
Rare Variants ◽  
Clinical Importance ◽  
Computational Prediction ◽  
Ethical Challenges ◽  
Inborn Errors ◽  
Rare Variation ◽  
Learning Healthcare System ◽  
The Impact

Genome sequencing is enabling precision medicine—tailoring treatment to the unique constellation of variants in an individual’s genome. The impact of recurrent pathogenic variants is often understood, leaving a long tail of rare genetic variants that are uncharacterized. The problem of uncharacterized rare variation is especially acute when it occurs in genes of known clinical importance with functionally consequent frequent variants and associated mechanisms. Variants of unknown significance (VUS) in these genes are discovered at a rate that outpaces current ability to classify them using databases of previous cases, experimental evaluation, and computational predictors. Clinicians are thus left without guidance about the significance of variants that may have actionable consequences. Computational prediction of the impact of rare genetic variation is increasingly becoming an important capability. In this paper, we review the technical and ethical challenges of interpreting the function of rare variants in two settings: inborn errors of metabolism in newborns, and pharmacogenomics. We propose a framework for a genomic learning healthcare system with an initial focus on early-onset treatable disease in newborns and actionable pharmacogenomics. We argue that (1) a genomic learning healthcare system must allow for continuous collection and assessment of rare variants, (2) emerging machine learning methods will enable algorithms to predict the clinical impact of rare variants on protein function, and (3) ethical considerations must inform the construction and deployment of all rare-variation triage strategies, particularly with respect to health disparities arising from unbalanced ancestry representation.

Motor Coordination in Space: An Analysis of the Effects of Microgravity on XRCCI DNA Repair Protein Function

2020 ◽  
Author(s):  
Vishruth Nagam
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Function ◽  
Motor Coordination ◽  
Strand Break ◽  
Single Strand Break ◽  
Single Strand Break Repair ◽  
Repair Protein ◽  
Mature Neurons ◽  
Microgravity Conditions

Abstract While in space, astronauts have been known to face exposure to stressors that may increase susceptibility to DNA damage. If DNA repair proteins are defective or nonexistent, DNA mutations may accumulate, causing increasingly abnormal function as one ages [1]. The DNA single-strand break repair protein XRCC1 is important for cerebellar neurogenesis and interneuron development [2]. According to previous studies, a deficiency of XRCC1 can lead to an increase in DNA damage, in mature neurons, and ataxia (a progressive loss of motor coordination) [2]. I propose to address how XRCC1’s efficiency can change in microgravity conditions. This experiment’s relevance is underscored by the importance of motor coordination and physical fitness for astronauts; determining the potential effects of microgravity on XRCC1 is crucial for future space exploration.

scholarly journals The impact of incomplete knowledge on evaluation: an experimental benchmark for protein function prediction

2009 ◽  
Vol 25 (18) ◽  
pp. 2404-2410 ◽  
Author(s):  
Curtis Huttenhower ◽  
Matthew A. Hibbs ◽  
Chad L. Myers ◽  
Amy A. Caudy ◽  
David C. Hess ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Function Prediction ◽  
Function Prediction ◽  
Incomplete Knowledge ◽  
The Impact

scholarly journals A homozygous telomerase T-motif variant resulting in markedly reduced repeat addition processivity in siblings with Hoyeraal Hreidarsson syndrome

Blood ◽  
2013 ◽  
Vol 121 (18) ◽  
pp. 3586-3593 ◽  
Author(s):  
Maria M. Gramatges ◽  
Xiaodong Qi ◽  
Ghadir S. Sasa ◽  
Julian J.-L. Chen ◽  
Alison A. Bertuch
Keyword(s):  
Protein Function ◽  
Severe Disease ◽  
Computational Algorithms ◽  
Key Points ◽  
The Impact ◽  
Motif Variant ◽  
Limited Sensitivity

Key Points Biallelic inheritance of a telomerase T-motif mutation selectively impairs repeat addition processivity and results in severe disease. Computational algorithms commonly used to predict the impact of variants on protein function have limited sensitivity with regard to hTERT.

scholarly journals Evaluating the role of RAD52 and its interactors as novel potential molecular targets for hepatocellular carcinoma

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Ping Li ◽  
YanZhen Xu ◽  
Qinle Zhang ◽  
Yu Li ◽  
Wenxian Jia ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dna Repair ◽  
Characteristic Curve ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Protein Structure Analysis ◽  
Qrt Pcr ◽  
Specificity And Sensitivity ◽  
Huh7 Cells ◽  
The Impact

Abstract Background Radiation sensitive 52 (RAD52) is an important protein that mediates DNA repair in tumors. However, little is known about the impact of RAD52 on hepatocellular carcinoma (HCC). We investigated the expression of RAD52 and its values in HCC. Some proteins that might be coordinated with RAD52 in HCC were also analyzed. Methods Global RAD52 mRNA levels in HCC were assessed using The Cancer Genome Atlas (TCGA) database. RAD52 expression was analyzed in 70 HCC tissues and adjacent tissues by quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry. The effect of over-expressed RAD52 in Huh7 HCC cells was investigated. The String database was then used to perform enrichment and functional analysis of RAD52 and its interactome. Cytoscape software was used to create a protein–protein interaction network. Molecular interaction studies with RAD52 and its interactome were performed using the molecular docking tools in Hex8.0.0. Finally, these DNA repair proteins, which interact with RAD52, were also analyzed using the TCGA dataset and were detected by qRT-PCR. Based on the TCGA database, algorithms combining ROC between RAD52 and RAD52 interactors were used to diagnose HCC by binary logistic regression. Results In TCGA, upregulated RAD52 related to gender was obtained in HCC. The area under the receiver operating characteristic curve (AUC) of RAD52 was 0.704. The results of overall survival (OS) and recurrence-free survival (RFS) indicated no difference in the prognosis between patients with high and low RAD52 gene expression. We validated that RAD52 expression was increased at the mRNA and protein levels in Chinese HCC tissues compared with adjacent tissues. Higher RAD52 was associated with older age, without correlation with other clinicopathological factors. In vitro, over-expressed RAD52 significantly promoted the proliferation and migration of Huh7 cells. Furthermore, RAD52 interactors (radiation sensitive 51, RAD51; X-ray repair cross complementing 6, XRCC6; Cofilin, CFL1) were also increased in HCC and participated in some biological processes with RAD52. Protein structure analysis showed that RAD52–RAD51 had the firmest binding structure with the lowest E-total energy (− 1120.5 kcal/mol) among the RAD52–RAD51, RAD52–CFL1, and RAD52–XRCC6 complexes. An algorithm combining ROC between RAD52 and its interactome indicated a greater specificity and sensitivity for HCC screening. Conclusions Overall, our study suggested that RAD52 plays a vital role in HCC pathogenesis and serves as a potential molecular target for HCC diagnosis and treatment. This study’s findings regarding the multigene prediction and diagnosis of HCC are valuable.

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