Single-cell splicing analysis of the heart in myotonic dystrophy

项目总结/摘要 强直性肌营养不良1型（DM 1）是一种高度可变的遗传性疾病，具有不可预测的表现， 严重程度和多系统症状的进展，这些症状在个体之间可能差异很大，甚至 在同一个家庭的成员之间。DM 1是由DMPK基因中的CTG重复扩增引起的， 转录成具有长CUG重复序列的RNA，其结合并隔离前mRNA的重要调节因子 拼接因此，DM 1的分子标志是一组基因的错误剪接，这些基因有助于 疾病表型CTG重复扩增也是高度不稳定的，并随着时间的推移继续扩增 在DM 1个体中，以不同的速率，导致高度的体细胞镶嵌，我们 假设有助于这种进行性疾病的症状变异性。此外，高达80%的 患有DM 1的个体具有导致危及生命的心律失常和心源性猝死的心脏缺陷， 占该病死亡人数的30%。然而，大多数关于心脏的选择性剪接研究， DM 1的特征仅在小鼠模型上进行。为了确定是否发现了错误剪接事件， 在小鼠模型中，这些基因在人类中是保守的，并且具有类似的功能后果，需要更多的研究。 临床相关的样本。 这个项目的总体目标是利用大转录组数据与单细胞分辨率沿着 假设驱动的研究，使用分子和基因组工具来研究疾病机制， DM 1的治疗方法单细胞RNA测序（scRNA-seq）将在人诱导的 来自DM 1患者和来自未受影响的DM 1患者的多能干细胞衍生的心肌细胞（iPSC-CM） 个体然后将使用最新的生物信息学方法分析生成的数据， iPSC-CM中选择性剪接的全转录组研究，并揭示了细胞镶嵌和心脏 DM 1的发病机制此外，将设计反义寡核苷酸以纠正错误剪接事件 并确定其对DM 1 iPSC-CM中疾病表型的贡献。潜在的假设是， 新的致病性错误剪接事件可以从DM 1 iPSC-CM的scRNA-seq中鉴定， 在批量RNA测序中被掩盖，调节这些错误剪接事件可以帮助挽救心脏病。 表型该项目的结果将：1）确定iPSC的单细胞选择性剪接谱- CMs，2）阐明DM 1中体细胞嵌合现象与表型变异之间的联系，3）鉴定 DM 1中的致病性错误剪接事件，和4）揭示了纠正有害错误剪接的潜在方法。

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.