Discovering cardiomyopathy modifiers via zebrafish genetics

 描述(申请人提供)：心肌病是一组具有遗传异质性和表达多样性的疾病。为了开发有效的治疗方法，识别致病基因和遗传修饰因素是至关重要的。这项建议旨在建立一种新的基于成年斑马鱼的筛查方法，允许系统地识别心肌病的遗传修饰因素。在建立了阿霉素诱导的心肌病模型并开发了一种产生心脏突变的有效方法后，我们为此目的测试了一种正向遗传筛选方法。对&GT；500基因破碎型转座子(GBT)突变体进行的中试筛选确定了四个突变株，它们可以改变DOX诱导的心肌病。三个受影响的基因，包括sorbin和SH3结构域包含2b的sorbs2b(Sorbs2b)，anoctamin 5a(Ano5a)和维甲酸X受体，αa(Rxraa)，先前被认为与心肌病有关；而第四个基因，DNAJ(Hsp40)同源物，B亚家族，成员b(Dnajb6b)，是一个新的心脏基因，表明我们的筛选方法有能力发现已知和新的心肌病基因。在我们放大屏幕之前，我们认为确定这些斑马鱼突变是否以及如何促进我们对人类心肌病的理解是至关重要的。通过关注新的心脏基因dnajb6b和新定义的非心肌细胞表达的基因sorbs2b，我们建议检验这一建议的中心假设，预测可以通过突变筛选方法识别新的心肌病修饰基因，可以进一步研究这些基因对不同遗传类型心肌病的修饰作用，并开发靶向治疗。该提案分为以下三个具体目标。在具体目标1中，我们将确定dnajb6b和sorbs2b对不同类型遗传性心肌病的修饰作用。为此，我们建立了三种遗传性心肌病模型，包括bag3 KO、mBAG3 OE和LMNA KO。在具体目标2中，我们将通过机制研究阐明dna jb6b和sorbs2b的修饰作用，并寻求治疗方法。我们将研究抑制UPR是否改善dnajb6b和/或sorbs2b的修饰效果。在具体目标3中，我们将在鱼类和小鼠模型上验证人DNAJB6和SORBS2突变体的修饰效果以及靶向治疗。这些目标的完成有望证明斑马鱼用于建立不同类型心肌病模型的可行性，识别修饰基因，阐明这些基因对不同类型心肌病的修饰作用，并发现靶向治疗方法。如果成功，我们的策略将极大地推进心肌病的预后测试开发、风险分层和个性化治疗。

 DESCRIPTION (provided by applicant): Cardiomyopathy is a group of disorder with genetic heterogeneity and variable expression. To develop effective therapy, it is vital to identify both causative genes and genetic modifiers. This proposal aims to establish a novel adult zebrafish-based screening approach that allows systematically identification of genetic modifiers of cardiomyopathy. Having generated a Doxorubicin-induced cardiomyopathy model and developed an efficient method to generate cardiac mutants, we tested a forward genetic screening approach for this purpose. A pilot screen of >500 gene-breaking transposon (GBT) mutants identified four mutant lines that modified DOX-induced cardiomyopathy. Three affected genes, including sorbin and SH3 domain containing 2b (sorbs2b), anoctamin 5a (ano5a) and retinoid X receptor, alpha a (rxraa), have been previously linked to cardiomyopathy; while the fourth gene, Dnaj (Hsp40) homolog, subfamily B, member b (dnajb6b), is a new cardiac gene, demonstrating the capacity of our screening method to discover both known and new cardiomyopathy genes. Before we scale up the screen, we consider it as pivotal to determine whether and how these zebrafish mutants facilitate our understanding of human cardiomyopathy. By focusing on dnajb6b, the new cardiac gene, and sorbs2b, the gene with newly defined non-cardiomyocyte expression, we propose to test the central hypothesis of this proposal predicting that novel cardiomyopathy modifying genes can be identified via a mutagenesis screen approach, which can be further studied to profile their modifying effects on different genetic types of cardiomyopathy and to develop targeted therapy. The proposal is divided into the following three specific aims. In Specific Aim 1, we will determine the modifying effects of dnajb6b and sorbs2b on different types of heritable cardiomyopathy. For this purpose, we have generated three heritable cardiomyopathy models including bag3 KO, mBAG3 OE and lmna KO. In Specific Aim 2, we will elucidate the modifying effects of dnajb6b and sorbs2b via mechanistic studies and to seek therapeutics. We will examine whether UPR inhibition ameliorates the modifying effects of dnajb6b and/or sorbs2b. In specific Aim 3, we will validate the modifying effects of human DNAJB6 and SORBS2 variants and the targeted therapeutics in both fish and mouse models. Completion of these aims are expected to demonstrate the feasibility of zebrafish to model different types of cardiomyopathy, to identify modifying genes, t elucidate the modifying effects of these genes on different type of cardiomyopathies, and to discover targeted therapeutics. If successful, our strategy shall greatly advance prognostic test development, risk stratification, and personalized therapy for cardiomyopathy.