Yamamoto Yuta, Chua Kaiser, Ferrasse Alexis, Kirilova Anna, De Jong Hannah N, Floyd Brendan J, Cadisch Christian, Wiel Laurens, Wang Qianru, O'Neill Matthew J, Tabet Daniel, Staudt David, Goryznski John E, Huang Yong, Wilson Rachel H, Sharma Arman, Tapales Althea, Agrawal Rani, Wheeler Matthew T, MacRae Calum, Roden Dan M, Roth Frederick P, Glazer Andrew M, Ashley Euan A, Parikh Victoria N
Stanford Center for Inherited Cardiovascular Disease, Division of Cardiovascular Medicine, Department of Medicine, Stanford School of Medicine, Palo Alto, CA.
Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, PA, USA.
bioRxiv. 2025 May 27:2025.05.23.655878. doi: 10.1101/2025.05.23.655878.
An estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 () gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms.
We developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).
Our multidimensional mapping strategy enabled high-resolution functional analysis of variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants.
This work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.
据估计，每500人中就有1人患有肥厚型心肌病（HCM），这种疾病的基因诊断可以识别有风险的家庭成员，并越来越多地指导治疗。肌球蛋白结合蛋白C3（MYBPC3）基因突变占HCM病例的很大比例。然而，这些变异中的许多被归类为意义未明的变异（VUS），这使临床决策变得复杂。在疾病特异性细胞类型中进行变异解读的可扩展方法对于理解变异影响和揭示疾病机制至关重要。
我们开发了一种规模化的多维映射策略，以评估MYBPC3关键结构域上变异的功能影响。我们在天然MYBPC3位点进行饱和碱基编辑，这是一种基于长读长RNA测序的变异剪接效应检测方法，并测量与HCM相关的表型，包括人诱导多能干细胞衍生的心肌细胞（iPSC-CMs）中MYBPC3丰度、肥厚信号传导和泛素-蛋白酶体功能。
我们的多维映射策略能够在iPSC-CMs中对变异进行高分辨率功能分析。靶向瞬时碱基编辑在天然位点生成了一个全面的变异文库，捕获了对细胞HCM相关表型的各种变异效应。我们的大规模平行剪接检测鉴定出了新的剪接破坏变异。功能检测的整合表明，MYBPC3丰度降低是HCM相关表型的关键驱动因素。同时，观察到蛋白质降解下调是对MYBPC3功能丧失的一种代偿反应，并为关键结合域附近的错义变异确定了新的疾病机制，强调了它们对发病机制的贡献。变异效应的贝叶斯估计能够对临床变异进行重新分类。
这项工作提供了一个平台，可将iPSC中的基因组工程扩展到对多种与疾病相关的细胞表型的变异效应进行多重检测，增强对变异致病性的理解，并揭示可指导治疗策略的新生物学机制。
