Genetic Studies of Sarcomere-based Cardiac Diseases (Diversity Supplement)

已发现肌联蛋白（TTN）截短变体（TTNtvs）是最常见的遗传因子， 扩张型心肌病（DCM）。然而，TTNtv DCM（即TTNtv）的等位基因异质性（AH）也是 在参考人群中发现，显著混淆了这些疾病的诊断和治疗开发 患者致病性TTNtv主要存在于C末端A带区（TTNtv-As），但在 N-末端Zdisc区域（TTNtv-Zs）。此外，TTNtv DCM的病理信号通路仍然存在， 大部分未知。在这里，我们的目标是利用斑马鱼遗传学带来的独特研究机会 破译AH的潜在机制，发现病理信号通路，并开发 有效的治疗方法。我们的初步研究表明TTNtv DCM的AH可以 在胚胎和成年斑马鱼中重现，为AH的机制研究打开了大门， vivo.从我们对已知心肌病信号通路的筛选中，我们确定了mTOR，自噬， MAPK和PDE 1作为候选信号通路，可用于治疗益处。我们 还建立了一个基于F0的遗传分析，使用微同源介导的末端连接（MMEJ） 基因组编辑技术使我们能够快速发现新的信号通路。基于这些 初步研究，我们建议利用斑马鱼独特的遗传和化学遗传工具， 证明斑马鱼是TTNtv DCM等位基因异质性的第一个体内动物模型， 用于破译TTNtv引起的原发性损伤，以发现连续的病理信号传导 途径，并开发基于机制的疗法。该提案分为三个具体的 目标。在具体目标1中，我们将通过研究ttntv基因组来解读ttntv DCM的等位基因异质性。 突变体和TTN无效突变体。在具体目标2，我们建议阐明自噬的分子基础 在ttntv DCM中的失调，并开发基于自噬的治疗。在具体目标3中，我们将确认 MAPK和PDE 1作为候选信号通路，并发现其他新的基因和信号通路， 携带基于MMEJ的F0屏幕。在建议完成后，我们预计 可交付成果：1）提供体内证据以阐明TTNtv-As更可能导致DCM的原因 表型比TTNtv-Zs; 2）获得ttntv中自噬，MAPK和PDE信号通路的见解 DCM，并确定ttntv DCM的基于机制的治疗途径; 3）建立基于F0的遗传 一种能够系统地发现ttntv DCM新基因的筛选方法， 遗传性心肌病机制研究的前所未有的机会

TITIN (TTN) truncating variants (TTNtvs) have been found to be the most common genetic factor for dilated cardiomyopathy (DCM). However, allelic heterogeneity (AH) of TTNtv DCM, i.e. TTNtvs are also found in reference populations, significantly confound diagnosis and therapeutic development of these patients. Pathogenic TTNtvs are mainly found in the C-terminal A-band region (TTNtv-As) but less in the N-terminal Zdisc region (TTNtv-Zs). Moreover, pathological signaling pathways for TTNtv DCM remain largely unknown. Here, we aim to leverage unique research opportunities enabled by zebrafish genetics to decipher underlying mechanisms of AH, discover pathological signaling pathways, and develop effective therapeutic avenues. Our preliminary studies showed that AH of TTNtv DCM can be recapitulated in both embryonic and adult zebrafish, opening the door for mechanistic studies of AH in vivo. From our screen of known cardiomyopathy signaling pathways, we identified mTOR, autophagy, MAPK and PDE1 as candidate signaling pathways that could be leveraged for therapeutic benefits. We also established a F0-based genetic assay using the Microhomologymediated end joining (MMEJ) genome editing technology that enables us to rapidly discover new signaling pathways. Based on these preliminary studies, we proposed to leverage unique genetic and chemical genetic tools in zebrafish to prove that zebrafish is the first in vivo animal model for allelic heterogeneity of TTNtv DCM, which can be used to decipher primary damages incurred by TTNtvs, to discover sequential pathological signaling pathways, and to develop mechanism-based therapies. The proposal is organized into the 3 specific aims. In Specific Aim 1, we will decipher allelic heterogeneity of ttntv DCM via studying a panel of ttntv mutants and ttn null mutants. In Specific Aim 2, we propose to elucidate molecular basis of autophagy dysregulation in ttntv DCM and develop an autophagy-based therapy. In Specific Aim 3, we will confirm MAPK and PDE1 as candidate signalings and discover additional new genes and signaling pathways by carrying MMEJ-based F0 screens. Upon completion of the proposal, we anticipate the following deliverables: 1) provide in vivo evidence to clarify why TTNtv-As are more likely to cause DCM phenotypes than TTNtv-Zs; 2) obtain insights on autophagy, MAPK and PDE signaling pathways in ttntv DCM, and identify mechanism-based therapeutic avenues for ttntv DCM; 3) establish a F0-based genetic screening approach that is capable of systematically discovering new genes for ttntv DCM, opening an unprecedented opportunity for mechanistic studies of an inherited cardiomyopathy