PDF(Pubmed)
Abstract：
Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.
摘要：
星云,骨骼肌细丝的关键蛋白质，在调节细丝长度(TFL)等生理过程中起着重要作用，跨桥自行车,和肌原纤维排列。星状蛋白基因（NEB）的致病变异导致基于NEB的线虫肌病（NEM2），一种以张力减退和肌肉无力为特征的遗传异质性疾病，目前缺乏治疗。在这项研究中,我们检查了10名NEM2患者，每个都有独特的致病变异，为了了解它们对mRNA的影响，蛋白质，和功能层面。结果显示致病性截短变体影响NEBmRNA稳定性并导致突变转录物的无义介导的衰变。此外，在具有致病性剪接变异体的患者中发现隐性剪接位点激活的发生率很高,这些变异体预期会破坏星云蛋白的肌动蛋白结合位点.蛋白质水平的测定显示，星云蛋白相对正常或明显减少的患者。我们观察到星云的减少和TFL的减少之间存在正相关关系，或减少张力（最大和次最大张力）。有趣的是,我们的研究揭示了星云中的致病性重复变异，导致NEB一式三份区域获得四拷贝，并且星云蛋白蛋白更大，TFL更长。此外,我们调查了Omecamtivmecarbil(OM)的效果，一种小分子心肌肌球蛋白激活剂,对NEM2患者1型肌纤维产生力的影响。OM治疗显著增加了所有NEM2患者的亚最大张力，范围从87％到318％，在星云蛋白水平最低的患者中效果最大。总之,这项研究表明，转录后或翻译后机制调节星云蛋白的表达。此外，我们认为NEM2的病理机制不仅涉及缩短，而且涉及细长的细丝，伴随着致病性剪接变体导致的肌动蛋白结合位点的破坏。重要的是,我们的发现强调了OM治疗改善NEM2患者骨骼肌功能的潜力，尤其是那些星云水平大幅下降的人。
