Pathogenic mechanisms of myosin binding protein C missense variants within hypertrophic cardiomyopathy

项目摘要/摘要 肥厚型心肌病(HCM)是一种常见的遗传性疾病，影响约1：500人， 严重的临床负担，包括心力衰竭、心律失常和心脏性猝死。最常见的 受影响的蛋白是肌球蛋白结合蛋白C3(MYBPC3)。将MYBPC3变异定义为致病使能 高危亲属的临床预测和基因筛查。截断MYBPC3的变体会导致 过早终止密码子导致功能丧失。然而，MYBPC3中的误解变体，通常被归类为 意义不确定的变体很难解释，因为蛋白质可能会容忍一种氨基的取代 另一种是酸。先前的细胞和结构数据表明致病错义变异可能导致肥厚型心肌梗死 通过两种不同的机制。一些致病错义变体通常会整合到肌原纤维和DR中。 汤普森假设这些变体通过抑制蛋白质结合而导致肥厚性心肌梗死。要评估这一点 假设，在目标1中，汤普森博士将研究致病错义引起的蛋白质结合的变化。 变种使用亲和质谱学。此外，她将描述患者衍生的细胞模型和 最常见的致病错义变体的敲入小鼠模型预计将通过这一机制发挥作用 发病机制为R502W，占HCM病例的2.4%。这些模型将评估HCM表型 可以被野生型MYBPC3的表达逆转，这表明类似的靶向治疗方法可以 用于这种常见的错义变量和截断变量。其他致病错义变体包括 迅速从细胞中清除，类似于截断变体，汤普森博士假设这些变体 通过热力学不稳定性导致HCM。支持这一假设的是，她之前的计算工作 证明了患有肥厚型心肌炎和MYBPC3 VU的患者经计算预测会导致亚域 错误折叠显示出更高的不良临床结局，类似于肥厚型心肌炎患者和已知的 MYBPC3致病变异株。在目标2中，她将评估是否采用循序渐进的实验方法， 首先在各个子域中以高吞吐量的方式描述变体，然后在 使用全长MYBPC3的心肌细胞将能够加速识别导致 蛋白质错误折叠。综上所述，这项工作应该为致病机理提供新的生物学见解。 MYBPC3错义变异的机制(S)，使肥厚性心肌病患者的临床预后得到改善 和高危亲属，并告知治疗肥厚型心肌炎的个性化靶向治疗方法。这项建议是 有别于汤普森博士以前在化学生物学方面的经验，这将为她提供宝贵的 心肌病的疾病模型、心脏生理学和高级实验遗传学。这份工作，她 导师团队，以及所描述的培训活动将促进汤普森博士对她的发展 最终目标是成为一名学术内科医生-科学家和独立调查者。

PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) is a common genetic disease, affecting ~ 1:500 people, with substantial clinical burden including heart failure, arrhythmia and sudden cardiac death. The most commonly affected protein is myosin binding protein C3 (MYBPC3). Defining a MYBPC3 variant as pathogenic enables clinical prognostication and genetic screening in at-risk relatives. Truncating variants of MYBPC3 cause a premature stop codon resulting in loss of function. However, missense variants in MYBPC3, often classified as variants of uncertain significance, are difficult to interpret as proteins may tolerate the substitution of one amino acid for another. Prior cellular and structural data suggest that pathogenic missense variants may lead to HCM via two distinct mechanisms. Some pathogenic missense variants normally incorporate into myofibrils and Dr. Thompson hypothesizes that these variants lead to HCM via inhibition of protein binding. To evaluate this hypothesis, in aim 1, Dr. Thompson will study alterations in protein binding caused by pathogenic missense variants using affinity mass spectrometry. Further, she will characterize a patient-derived cellular model and a knock-in mouse model of the most common pathogenic missense variant predicted to work via this mechanism, R502W, which accounts for 2.4% of HCM cases. These models will evaluate if HCM phenotypes can be reversed by expression of wild-type MYBPC3, suggesting a similar targeted therapeutic approach can be utilized for this common missense variant and truncating variants. Other pathogenic missense variants are rapidly cleared from the cell, similar to truncating variants, and Dr. Thompson hypothesizes that these variants lead to HCM via thermodynamic instability. Supporting this hypothesis, her prior computational work demonstrated that patients with HCM and a MYBPC3 VUS computationally predicted to cause subdomain misfolding exhibited higher rates of adverse clinical outcomes, similar to patients with HCM and known MYBPC3 pathogenic variant. In aim 2, she will evaluate if a step-wise experimental approach, which characterizes variants first in a high-throughput manner within individual subdomains and then within cardiomyocytes using full-length MYBPC3, will enable accelerated identification of variants which cause protein misfolding. Taken together, this work should provide novel biologic insight into the pathogenic mechanism(s) of MYBPC3 missense variants, enable improved clinical prognostication for patients with HCM and at-risk relatives, and inform personalized targeted therapeutic approaches to treat HCM. This proposal is distinct from Dr. Thompson’s prior experience in chemical biology and will provide her with valuable training in disease models of cardiomyopathy, cardiac physiology, and advanced experimental genetics. This work, her mentorship team, and the training activities described will facilitate Dr. Thompson’s development toward her ultimate goal of becoming an academic physician-scientist and independent investigator.