Characterization of TTN splicing variants to gain further insights into their role in skeletal muscle disorders

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Title: Characterization of TTN splicing variants to gain further insights into their role in skeletal muscle disorders
Author: Qureshi, Talha
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Faculty of Biological and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2019
Language: eng
Thesis level: master's thesis
Discipline: perinnöllisyystiede
Abstract: The TTN gene encodes a giant muscle protein called titin that regulates the function of muscle sarcomere and interacts with several other muscle proteins. Mutations in TTN are associated with a broad range of skeletal and cardiac muscle disorders termed titinopathies. Previous studies have shown the importance of unusual TTN splicing events in patients with TTN-related cardiomyopathies and muscular dystrophies. In this project, we characterized eight TTN splicing variants to further expound on the pathogenesis of titinopathies and to enhance the diagnostic accuracy for patients with TTN mutations. In addition, we also made a comparative analysis of five different RNA/cDNA sequencing techniques to extrapolate on which approach is most suitable to study splicing variants in TTN gene. Skeletal muscle samples of six patients were analyzed in this study who were previously detected with TTN variants in a compound heterozygous state from a targeted next-generation sequencing assay. Our results from traditional Sanger sequencing methods, second-generation (Illumina RNA-Sequencing) and third-generation sequencing (Single-molecule real-time sequencing) methods showed distinct splicing events in the form of partial or complete exon skipping, intron retention, and in few instances showed multiple splicing effects rendered by a single variant. Complying with the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, the splicing variants were classified as pathogenic, likely pathogenic or variant of uncertain significance primarily on the basis of our experimental data. To address which sequencing method is most promising for analyzing TTN splicing variants, Illumina RNA Sequencing is very efficient, though, the combination of Illumina RNA Sequencing with long-read sequencing could be ideal. Our results further demonstrate that a near full-length titin is vital for survival until birth, and further studies are needed to understand the pathophysiology mechanism of congenital titinopathies.
Subject: alternative splicing

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