Single-cell splicing analysis of the heart in myotonic dystrophy

项目摘要/摘要 强直性肌营养不良1型(DM1)是一种高度可变的遗传性疾病，具有不可预测的表现， 多系统症状的严重程度和进展，这些症状在个体之间甚至是 在同一个家庭的成员之间。DM1是由DMPK基因中CTG重复序列的扩张引起的 转录成带有长CUG重复序列的RNA，该重复序列与Pre-mRNA的重要调节因子结合并隔离 拼接。因此，DM1的一个分子特征是导致致病基因子集的错误拼接 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张 在一个患有DM1的个体内以不同的比率，导致高度的体细胞嵌合体，我们 假说有助于这种进行性疾病的症状可变性。此外，高达80%的 患有DM1的人有心脏缺陷，会导致危及生命的心律失常和心脏性猝死， 占该病总死亡率的30%。然而，大多数关于心脏的选择性剪接研究 DM1的功能仅在小鼠模型上完成。确定是否发现错误拼接事件 小鼠模型在人类中是保守的，并且具有类似的功能后果，还需要更多的研究 与临床相关的样本。 该项目的总体目标是利用具有单细胞分辨率的大型转录数据 通过使用分子和基因组工具进行假设驱动的研究来研究疾病机制和 DM1的治疗方法。单细胞rna测序(scrna-seq)将在人诱导的 DM1患者和非DM1患者多能干细胞来源的心肌细胞 个人。然后将使用最新的生物信息学方法分析生成的数据，以生成 IPSC-CMS中选择性剪接的转录组研究和揭示细胞嵌合体和心脏 DM1的发病机制。此外，反义寡核苷酸将被设计用来纠正错误剪接事件 并确定其在DM1 IPSC-CMS中对疾病表型的贡献。基本的假设是 新的致病错接事件可以从DM1 IPSC-CMS的scRNA-seq中识别出来 在大量RNA测序中被掩盖并调节这些错误剪接事件有助于挽救心脏 表型。该项目的结果将：1)确定IPSC的单细胞选择性剪接图谱- CMS，2)阐明体细胞嵌合体与DM1表型变异的关系，3)鉴定 DM1中的致病性错误剪接事件，以及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.