Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA; Center for Cardiovascular Research, Division of Cardiology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
J Mol Cell Cardiol. 2023 Mar;176:58-67. doi: 10.1016/j.yjmcc.2023.01.014. Epub 2023 Feb 3.
Dilated cardiomyopathy (DCM) is a leading cause of heart failure and a major indicator for heart transplant. Human genetic studies have identified over a thousand causal mutations for DCM in genes involved in a variety of cellular processes, including sarcomeric contraction. A substantial clinical challenge is determining the pathogenicity of novel variants in disease-associated genes. This challenge of connecting genotype and phenotype has frustrated attempts to develop effective, mechanism-based treatments for patients. Here, we identified a de novo mutation (T237S) in TPM1, the gene that encodes the thin filament protein tropomyosin, in a patient with DCM and conducted in vitro experiments to characterize the pathogenicity of this novel variant. We expressed recombinant mutant protein, reconstituted it into thin filaments, and examined the effects of the mutation on thin filament function. We show that the mutation reduces the calcium sensitivity of thin filament activation, as previously seen for known pathogenic mutations. Mechanistically, this shift is due to mutation-induced changes in tropomyosin positioning along the thin filament. We demonstrate that the thin filament activator omecamtiv mecarbil restores the calcium sensitivity of thin filaments regulated by the mutant tropomyosin, which lays the foundation for additional experiments to explore the therapeutic potential of this drug for patients harboring the T237S mutation. Taken together, our results suggest that the TPM1 T237S mutation is likely pathogenic and demonstrate how functional in vitro characterization of pathogenic protein variants in the lab might guide precision medicine in the clinic.
扩张型心肌病（DCM）是心力衰竭的主要原因，也是心脏移植的主要指征。人类遗传研究已经在涉及多种细胞过程的基因中发现了超过 1000 种导致 DCM 的因果突变，包括肌节收缩。一个重大的临床挑战是确定疾病相关基因中新变体的致病性。这种连接基因型和表型的挑战，使我们难以针对患者开发出有效、基于机制的治疗方法。在这里，我们在一名 DCM 患者中发现了 TPM1 基因（编码细丝蛋白原肌球蛋白的基因）中的一个新生突变（T237S），并进行了体外实验来描述该新突变的致病性。我们表达了重组突变蛋白，将其重新组装成细丝，并检查了该突变对细丝功能的影响。我们发现该突变降低了细丝激活的钙敏感性，这与先前已知的致病性突变所观察到的情况一致。从机制上讲，这种转变是由于突变诱导的原肌球蛋白在细丝上的位置变化引起的。我们证明，细丝激活剂 omecamtiv mecarbil 恢复了由突变原肌球蛋白调节的细丝的钙敏感性，这为进一步探索该药物对携带 T237S 突变的患者的治疗潜力奠定了基础。总之，我们的结果表明，TPM1 T237S 突变很可能是致病性的，并展示了如何在实验室中对致病性蛋白变体进行功能体外表征，从而指导临床精准医学。
