Dominant mutations in the calcium-permeable ion channel TRPV4 (transient receptor potential vanilloid 4) cause diverse and largely distinct channelopathies, including inherited forms of neuromuscular disease, skeletal dysplasias and arthropathy. Pathogenic TRPV4 mutations cause gain of ion channel function and toxicity that can be rescued by small molecule TRPV4 antagonists in cellular and animal models, suggesting that TRPV4 antagonism could be therapeutic for patients. Numerous variants in TRPV4 have been detected with targeted and whole exome/genome sequencing, but for the vast majority, their pathogenicity remains unclear. Here, we used a combination of clinical information and experimental structure-function analyses to evaluate 30 TRPV4 variants across various functional protein domains. We report clinical features of seven patients with TRPV4 variants of unknown significance and provide extensive functional characterization of these and an additional 17 variants, including structural position, ion channel function, subcellular localization, expression level, cytotoxicity and protein-protein interactions. We find that gain-of-function mutations within the TRPV4 intracellular ankyrin repeat domain target charged amino acid residues important for RhoA interaction, whereas ankyrin repeat domain residues outside of the RhoA interface have normal or reduced ion channel activity. We further identify a cluster of gain-of-function variants within the intracellular intrinsically disordered region that may cause toxicity via altered interactions with membrane lipids. In contrast, assessed variants in the transmembrane domain and other regions of the intrinsically disordered region do not cause gain of function and are likely benign. Clinical features associated with gain of function and cytotoxicity include congenital onset of disease, vocal cord weakness and motor-predominant disease, whereas patients with likely benign variants often demonstrated late-onset and sensory-predominant disease. These results provide a framework for assessing additional TRPV4 variants with respect to likely pathogenicity, which will yield critical information to inform patient selection for future clinical trials for TRPV4 channelopathies.

中文翻译：

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结合临床、结构和细胞研究区分致病性和良性 TRPV4 变异


钙渗透性离子通道 TRPV4（瞬时受体电位香草酸 4）的显性突变会导致多种多样且基本不同的离子通道病，包括遗传形式的神经肌肉疾病、骨骼发育不良和关节病。致病性 TRPV4 突变导致离子通道功能和毒性增加，在细胞和动物模型中可以通过小分子 TRPV4 拮抗剂来挽救，表明 TRPV4 拮抗剂可能对患者具有治疗作用。通过靶向和全外显子组/基因组测序已检测到 TRPV4 中的许多变异，但对于绝大多数变异，它们的致病性仍不清楚。在这里，我们结合使用临床信息和实验结构-功能分析来评估各种功能蛋白结构域的 30 个 TRPV4 变体。我们报告了 7 例意义未知的 TRPV4 变异患者的临床特征，并提供了这些变异和其他 17 种变异的广泛功能特征，包括结构位置、离子通道功能、亚细胞定位、表达水平、细胞毒性和蛋白质相互作用。我们发现 TRPV4 细胞内锚蛋白重复结构域内的功能获得性突变靶向对 RhoA 相互作用很重要的带电氨基酸残基，而 RhoA 界面外的锚蛋白重复结构域残基具有正常或降低的离子通道活性。我们进一步确定了细胞内固有无序区域内的一组功能获得性变异，这些变异可能通过改变与膜脂质的相互作用而引起毒性。相比之下，在跨膜结构域和固有无序区域的其他区域中评估的变异不会导致功能获得，并且可能是良性的。 与功能获得和细胞毒性相关的临床特征包括先天性疾病、声带无力和运动为主的疾病，而可能为良性变异的患者通常表现出晚发性和感觉为主的疾病。这些结果为评估其他 TRPV4 变体的可能致病性提供了一个框架，这将产生关键信息，为患者选择未来 TRPV4 通道病的临床试验提供信息。