Single-cell splicing analysis of the heart in myotonic dystrophy

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PROJECT SUMMARY/ABSTRACT Myotonic dystrophy type 1 (DM1) is a highly variable genetic disease with unpredictable manifestation, severity, and progression of multi-systemic symptoms that can vary dramatically between individuals and even across members of the same family. DM1 is caused by a CTG repeat expansion in the DMPK gene that are transcribed into RNA with long CUG repeats that bind to and sequester important regulators of pre-mRNA splicing. Consequently, a molecular hallmark of DM1 is the mis-splicing of a subset of genes that contribute to disease phenotypes. The CTG repeat expansions are also highly unstable and continues to expand over time at different rates within an individual with DM1, resulting in a high degree of somatic mosaicism that we hypothesize contributes to the symptomatic variability in this progressive disease. Furthermore, up to 80% of individuals with DM1 have cardiac defects that result in life-threatening arrhythmias and sudden cardiac death, composing up to 30% of all mortality in this disease. However, most alternative splicing studies on the cardiac features of DM1 have only been done on mouse models. To establish whether mis-splicing events discovered in mouse models are conserved in humans and with similar functional consequences, more studies are needed on clinically relevant samples. This overall goal of this project is to leverage big transcriptomic data with single-cell resolution along with hypothesis-driven research using molecular and genomic tools to investigate disease mechanisms and therapeutic methods for DM1. Single-cell RNA sequencing (scRNA-seq) will be done on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) derived from DM1 patients and from unaffected individuals. The data generated will then be analyzed using recent bioinformatical methods to generate a transcriptome-wide study of alternative splicing in iPSC-CMs and reveal the cellular mosaicism and cardiac pathogenesis in DM1. Additionally, antisense oligonucleotides will be designed to correct mis-splicing events and determine its contribution to disease phenotypes in DM1 iPSC-CMs. The underlying hypothesis is that novel pathogenic mis-splicing events can be identified from scRNA-seq of DM1 iPSC-CMs that have previously been masked in bulk RNA sequencing and modulating these mis-splicing events can help rescue cardiac phenotypes. The outcome of this project will: 1) determine the single-cell alternative splicing profile of iPSC- CMs, 2) elucidate the connection between somatic mosaicism and the phenotypic variability in DM1, 3) identify pathogenic mis-splicing events in DM1, and 4) reveal potential approaches to correct deleterious mis-splicing.