scholarly journals CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors

2019 ◽  
Author(s):  
Choongil Lee ◽  
Dong Hyun Jo ◽  
Gue-Ho Hwang ◽  
Jihyeon Yu ◽  
Jin Hyoung Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dna Sequence ◽  
Clinical Utility ◽  
Premature Termination ◽  
Genetic Disorders ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Potential Clinical Utility ◽  
Clinically Significant ◽  
Adenine Base

AbstractA nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read-through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.

scholarly journals Suppression of aminoglycoside-induced premature termination codon readthrough by the TRP channel inhibitor AC1903

2021 ◽  
Author(s):  
Alireza Baradaran-Heravi ◽  
Claudia C Bauer ◽  
Isabelle B Pickles ◽  
Sara Hosseini-Farahabadi ◽  
Aruna Balgi ◽  
...  
Keyword(s):  
Premature Termination ◽  
Genetic Disorders ◽  
Premature Termination Codon ◽  
Cation Channels ◽  
Trpc Channels ◽  
Nonsense Mutations ◽  
Variable Effect ◽  
Premature Termination Codons ◽  
Ic50 Values ◽  
Nonsense Mediated Mrna Decay

Nonsense mutations, which occur in ~11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as gentamicin and G418 permit PTC readthrough and so may address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we addressed the hypothesis that TRP non-selective cation channels contribute to the variable effect of aminoglycosides by controlling their cellular uptake. To attempt to identify the channel type involved, we tested AC1903, a 2-aminobenzimidazole derivative recently reported to selectively inhibit TRPC5 cation channels. AC1903 consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cell line and patient-derived JEB01 keratinocytes. In an effort to validate the suggested role of TRPC5, we tested an independent and more potent inhibitor called Pico145, which affects channels containing TRPC1, TRPC4 and TRPC5 but not other TRPCs or other channels. Unexpectedly, Pico145 was completely without effect, suggesting that AC1903 may work through other or additional targets. Consistent with this suggestion, AC1903 inhibited multiple TRPC channels including homomeric TRPC3, TRPC4, TRPC5, TRPC6 as well as concatemeric TRPC4-C1 and TRPC5-C1 channels, all with low micromolar IC50 values. It also inhibited TRPV4 channels but had weak or no effects on TRPV1 and no effect on another non-selective cation channel, PIEZO1. Overall, our study reveals a suppressor of aminoglycoside-mediated PTC readthrough (i.e., AC1903) but suggests that this compound has previously unrecognised effects. These effects require further investigation to determine the molecular mechanism by which AC1903 suppresses aminoglycoside uptake and PTC readthrough.

scholarly journals Nonsense Mutations in the lsa-Like Gene in Enterococcus faecalis Isolates Susceptible to Lincosamides and Streptogramins A

2003 ◽  
Vol 47 (7) ◽  
pp. 2307-2309 ◽  
Author(s):  
Julia Dina ◽  
Brigitte Malbruny ◽  
Roland Leclercq
Keyword(s):  
Enterococcus Faecalis ◽  
Premature Termination ◽  
Clinical Isolates ◽  
Intrinsic Resistance ◽  
Nonsense Mutations ◽  
Active Efflux ◽  
Premature Termination Codons

ABSTRACT The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux. The lsa-like genes of two clinical isolates of E. faecalis susceptible to lincosamides and dalfopristin contained mutations that produced premature termination codons. Revertant mutants were obtained by selection on agar plates containing clindamycin.

scholarly journals Expanded Analysis of Secondary Germline Findings From Matched Tumor/Normal Sequencing Identifies Additional Clinically Significant Mutations

2019 ◽  
pp. 1-11 ◽  
Author(s):  
Ecaterina Ileana Dumbrava ◽  
Lauren Brusco ◽  
Molly S. Daniels ◽  
Chetna Wathoo ◽  
Kenna R. Shaw ◽  
...  
Keyword(s):  
Clinical Utility ◽  
Molecular Testing ◽  
Pathogenic Variants ◽  
Cancer Related Gene ◽  
Predisposition Genes ◽  
Potential Clinical Utility ◽  
Genetics And Genomics ◽  
Clinically Significant ◽  
Next Generation Sequencing Ngs ◽  
Secondary Results

Purpose Next-generation sequencing (NGS) for tumor molecular profiling can reveal secondary germline likely pathogenic and pathogenic variants (LPV/PV). The American College of Medical Genetics and Genomics (ACMG) recommends return of secondary results for a subset of 59 genes, but other genes with evidence of clinical utility are emerging. We previously reported that 4.3% of patients who underwent NGS of a targeted panel of 201 genes had LPV/PV on the basis of the ACMG list. We report the frequency of additional germline cancer-related gene variants and discuss their clinical utility. Patients and Methods Matched tumor and germline DNA NGS of a targeted panel of 201 genes was performed in a research laboratory on samples from 1,000 patients with advanced or metastatic solid tumors enrolled in a molecular testing protocol ( ClinicalTrials.gov identifier: NCT01772771). The frequency of germline LPV/PV in 54 cancer-related genes, beyond the genes in ACMG list, were analyzed. Results Among 1,000 patients who underwent tumor/normal DNA sequencing, 46 (4.6%) were found to have a germline LPV/PV in the following genes: AR (n = 5), ATM (n = 4), BAP1 (n = 1), CDH1 (n = 1), CDKN2A (n = 1), CHEK1 (n = 2), CHEK2 (n = 10), EGFR (n = 1), ERCC3 (n = 4), ERCC5 (n = 1), HNF1B (n = 1), HRAS (n = 1), MITF (n = 4), MLL3 (n = 1), NF1 (n = 3), PKHD1 (n = 4), PTCH1 (n = 1), and SMARCA4 (n = 1). Thus, 8.7% of patients had an LPV/PV, with two patients having two concomitant germline LPV/PV. Five mutations in high-penetrance hereditary cancer predisposition genes were selected to be returned to patients or their representatives: BAP1, CDH1, CDKN2A, EGFR, and SMARCA4. Conclusion Broader genomic testing is likely to identify additional secondary pathogenic germline alterations, some with potential clinical utility for return to patients and their relatives. The recommended genes for which germline results should be returned are continually changing, which warrants continued study.

scholarly journals Synergy between Readthrough and Nonsense Mediated Decay Inhibition in a Murine Model of Cystic Fibrosis Nonsense Mutations

2020 ◽  
Vol 22 (1) ◽  
pp. 344
Author(s):  
Daniel R. McHugh ◽  
Calvin U. Cotton ◽  
Craig A. Hodges
Keyword(s):  
Protein Function ◽  
Therapeutic Option ◽  
Genetic Disorders ◽  
Therapeutic Strategies ◽  
Primary Cells ◽  
Functional Protein ◽  
Nonsense Mutations ◽  
Nonsense Mediated Decay ◽  
Premature Termination Codons ◽  
Cftr Protein

Many heritable genetic disorders arise from nonsense mutations, which generate premature termination codons (PTCs) in transcribed mRNA. PTCs ablate protein synthesis by prematurely terminating the translation of mutant mRNA, as well as reducing mutant mRNA quantity through targeted degradation by nonsense-mediated decay (NMD) mechanisms. Therapeutic strategies for nonsense mutations include facilitating ribosomal readthrough of the PTC and/or inhibiting NMD to restore protein function. However, the efficacy of combining readthrough agents and NMD inhibitors has not been thoroughly explored. In this study, we examined combinations of known NMD inhibitors and readthrough agents using functional analysis of the CFTR protein in primary cells from a mouse model carrying a G542X nonsense mutation in Cftr. We observed synergy between an inhibitor of the NMD component SMG-1 (SMG1i) and the readthrough agents G418, gentamicin, and paromomycin, but did not observe synergy with readthrough caused by amikacin, tobramycin, PTC124, escin, or amlexanox. These results indicate that treatment with NMD inhibitors can increase the quantity of functional protein following readthrough, and that combining NMD inhibitors and readthrough agents represents a potential therapeutic option for treating nonsense mutations.

scholarly journals Engineered transfer RNAs for suppression of premature termination codons

2018 ◽  
Author(s):  
John D. Lueck ◽  
Jae Seok Yoon ◽  
Alfredo Perales-Puchalt ◽  
Adam L. Mackey ◽  
Daniel T. Infield ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Premature Termination ◽  
Translation Termination ◽  
Genetic Disorders ◽  
Ribosome Profiling ◽  
Inherited Disease ◽  
Transfer Rnas ◽  
Premature Termination Codons ◽  
Nonsense Codons

ABSTRACTPremature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within the cystic fibrosis transmembrane conductance regulator (CFTR).

Staring Down Death

Crisis ◽  
2016 ◽  
Vol 37 (3) ◽  
pp. 212-217 ◽  
Author(s):  
Thomas E. Joiner ◽  
Melanie A. Hom ◽  
Megan L. Rogers ◽  
Carol Chu ◽  
Ian H. Stanley ◽  
...  
Keyword(s):  
Suicide Risk ◽  
Clinical Utility ◽  
Blink Rate ◽  
Specific Task ◽  
Careful Planning ◽  
Eye Blink ◽  
Eye Blinks ◽  
Potential Clinical Utility ◽  
The Relationship

Abstract. Background: Lowered eye blink rate may be a clinically useful indicator of acute, imminent, and severe suicide risk. Diminished eye blink rates are often seen among individuals engaged in heightened concentration on a specific task that requires careful planning and attention. Indeed, overcoming one’s biological instinct for survival through suicide necessitates premeditation and concentration; thus, a diminished eye blink rate may signal imminent suicidality. Aims: This article aims to spur research and clinical inquiry into the role of eye blinks as an indicator of acute suicide risk. Method: Literature relevant to the potential connection between eye blink rate and suicidality was reviewed and synthesized. Results: Anecdotal, cognitive, neurological, and conceptual support for the relationship between decreased blink rate and suicide risk is outlined. Conclusion: Given that eye blinks are a highly observable behavior, the potential clinical utility of using eye blink rate as a marker of suicide risk is immense. Research is warranted to explore the association between eye blink rate and acute suicide risk.

Investigations into the Clinical Utility of Latex D-Dimer in the Diagnosis of Deep Venous Thrombosis

1993 ◽  
Vol 69 (01) ◽  
pp. 008-011 ◽  
Author(s):  
Cedric J Carter ◽  
D Lynn Doyle ◽  
Nigel Dawson ◽  
Shauna Fowler ◽  
Dana V Devine
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Lower Limb ◽  
Clinical Utility ◽  
Rapid Test ◽  
Fibrin Degradation Product ◽  
Current Form ◽  
Non Invasive ◽  
Clinically Significant ◽  
D Dimer

SummaryThe serial use of non-invasive tests has been shown to be a safe method of managing outpatients who are suspected of having lower limb deep venous thrombosis (DVT). Objective testing has shown that the majority of these outpatients do not have venous thrombosis. A rapid test to exclude DVT in these patients, without the need for expensive and inconvenient serial non-invasive vascular testing, would have practical and economic advantages.Studies measuring the fibrin degradation product D-dimer using enzyme-linked immunoassays (EIA) in patients with veno-graphically proven DVT suggest that it should be possible to exclude this condition by the use of one of the rapid latex bead D-dimer tests.We have examined 190 patients with suspected DVT using both a latex and an EIA D-dimer assay. The latex D-dimer test used in this study was negative in 7 of the 36 proven cases of DVT. This sensitivity of only 80% is not sufficient to allow this type of assay, in its current form, to be used as an exclusion test for DVT. The same plasma samples were tested with an EIA assay. This information was used to mathematically model the effects of selecting a range of D-dimer discriminant cut off points for the diagnosis of DVT. These results indicate that 62% of suspected clinically significant DVT could have this diagnosis excluded, with a 98% sensitivity, if the rapid latex or equivalent D-dimer test could be reformulated to measure less than 185 ng/ml of D-dimer.

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