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Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1.

ABSTRACT: Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1-MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery.

SUBMITTER: Garcia-Alcover I

PROVIDER: S-EPMC4213733 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Publications

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1.

García-Alcover Irma I Colonques-Bellmunt Jordi J Garijo Raquel R Tormo José R JR Artero Rubén R Álvarez-Abril Mari Carmen MC López Castel Arturo A Pérez-Alonso Manuel M

Disease models & mechanisms 20140919 11

Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding facto ...[more]

PMID: 25239918

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Project description:Importance:Life expectancy is greatly shortened in patients presenting with myotonic dystrophy type 1 (DM1), the most common neuromuscular disease. A reliable prediction of survival in patients with DM1 is critically important to plan personalized health supervision. Objective:To develop and validate a prognostic score to predict 10-year survival in patients with DM1. Design, Setting, and Participants:In this longitudinal cohort study, between January 2000 and November 2014, we enrolled 1296 adults referred to 4 tertiary neuromuscular centers in France for management of genetically proven DM1, including 1066 patients in the derivation cohort and 230 in the validation cohort. Data were analyzed from December 2016 to March 2017. Main Outcomes and Measures:Factors associated with survival by multiple variable Cox modeling, including 95% confidence intervals, and development of a predictive score validated internally and externally. Mean values are reported with their standard deviations. Results:Of the 1296 included patients, 670 (51.7%) were women, and the mean (SD) age was 39.8 (13.7) years. Among the 1066 patients (82.3%) in the derivation cohort, 241 (22.6%) died over a median (interquartile range) follow-up of 11.7 (7.7-14.3) years. Age, diabetes, need for support when walking, heart rate, systolic blood pressure, first-degree atrioventricular block, bundle-branch block, and lung vital capacity were associated with death. Simplified score points were attributed to each predictor, and adding these points yielded scores between 0 and 20, with 0 indicating the lowest and 20 the highest risk of death. The 10-year survival rate was 96.6% (95% CI, 94.4-98.9) in the group with 0 to 4 points, 92.2% (95% CI, 88.8-95.6) in the group with 5 to 7 points, 80.7% (95% CI, 75.4-86.1) in the group with 8 to 10 points, 57.9% (95% CI, 49.2-66.6) in the group with 11 to 13 points, and 19.4% (95% CI, 8.6-30.1) in the group with 14 points or more. In 230 patients (17.7%) included in the validation cohort, the 10-year survival rates for the groups with 0 to 4, 5 to 7, 8 to 10, 11 to 13, and 14 points or more were 99.3% (95% CI, 95.0-100), 80.6% (95% CI, 67.1-96.7), 79.3% (95% CI, 66.2-95.1), 43.2% (95% CI, 28.2-66.1), and 21.6% (95% CI, 10.0-46.8), respectively. The calibration curves did not deviate from the reference line. The C index was 0.753 (95% CI, 0.722-0.785) in the derivation cohort and 0.806 (95% CI, 0.758-0.855) in the validation cohort. Conclusions and Relevance:The DM1 prognostic score is associated with long-term survival.

Dataset Information

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1.

Publications

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1.

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