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

项目总结/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、心律失常和心源性猝死。最常见的 受影响的蛋白是肌球蛋白结合蛋白C3（MYBPC3）。将MYBPC3变体定义为致病性使得能够 在高危亲属中进行临床诊断和遗传筛查。MYBPC3的截短变体导致 提前终止密码子导致功能丧失。然而，MYBPC3中的错义变体，通常被归类为 不确定意义的变体，很难解释，因为蛋白质可以容忍一个氨基的取代， 酸为另一种。先前的细胞和结构数据表明致病性错义变异可能导致HCM 通过两种不同的机制。一些致病性错义变异体通常并入肌原纤维。 Thompson假设这些变异通过抑制蛋白结合导致HCM。评价这一 假设，在目标1中，汤普森博士将研究致病性错义引起的蛋白质结合的改变 使用亲和质谱分析变体。此外，她将表征患者来源的细胞模型和 最常见的致病性错义变体的敲入小鼠模型预测通过这种方式起作用。 机制，R502W，占HCM病例的2.4%。这些模型将评估HCM表型 可以通过表达野生型MYBPC3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体和截短变体。其他致病性错义变体是 迅速从细胞中清除，类似于截短变体，汤普森博士假设这些变体 通过热力学不稳定导致HCM。支持这一假设，她先前的计算工作 表明HCM患者和MYBPC3 VUS计算预测引起亚结构域 错误折叠表现出更高的不良临床结局率，与HCM患者相似， MYBPC3致病性变体。在目标2中，她将评估一种逐步的实验方法， 首先在单个子域内以高通量方式表征变体，然后在 使用全长MYBPC3的心肌细胞，将能够加速鉴定导致心肌细胞凋亡的变异体。 蛋白质错误折叠总之，这项工作应该提供新的生物学见解的致病性 MYBPC 3错义变体的机制，能够改善HCM患者的临床诊断 和有风险的亲属，并告知个性化的靶向治疗方法来治疗HCM。这项建议是 与汤普森博士先前在化学生物学方面的经验不同，将为她提供宝贵的培训， 心肌病、心脏生理学和先进实验遗传学的疾病模型。这项工作，她 导师团队，所描述的培训活动将促进汤普森博士的发展，她 最终目标是成为一名学术物理学家，科学家和独立调查员。

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.