Thick and Thin Filament Mutations that cause Distal Arthrogryposis

描述(申请人提供)：关节畸形是一种先天性肌挛缩症候群，每3000个活产儿中就有一个受到影响。虽然DA的遗传原因仍在发现中，但这些突变如何导致终生痉挛的生物物理机制仍不清楚。我们最近获得并报告了一例DA患者的收缩特征，显示出MYH3R672C突变。这种突变减少了人类骨骼肌活组织中单个皮肤肌纤维的稳态作用力产生，延长了松弛时间，我们的数据表明，这可能是通过改变交叉桥结合和循环动力学而发生的。这是如何发生的，其机制尚不清楚。因此，从包含细丝和粗丝肌节蛋白突变的患者活检中，可以从收缩评估中学到很多东西。我假设细丝蛋白突变和粗丝蛋白突变导致肌挛缩改变的方式是不同的。具体地说，我预计粗丝突变，如MYH3 R672C和MYH8 R672H，将导致肌球蛋白(交叉桥)结合、脱离和与肌动蛋白相互作用的直接变化。这些突变导致了一种严重的DA，弗里曼-谢尔顿综合征。相反，我预计细丝突变，如TNNI2 R174Q和TNNT3 R63H，将通过改变细丝激活的钙敏感性和协同性而导致交叉桥结合的变化。这些突变 导致一种轻微的DA形式，谢尔顿-霍尔综合征。通过确定功能变化和致病机制，我们可以开始开发靶向治疗，恢复分子和细胞水平的功能。我计划这样做有三个目标，重点是肌肉纤维和肌原纤维的肌肉收缩能力。我将使用肌肉纤维来确定最大力的产生、交叉桥的结合和力的钙敏感性。我将使用肌原纤维来确定从人类骨骼肌活检组织中分离的单个肌原纤维的细丝激活和松弛动力学。最后，我将使用肌肉纤维和潜在的肌原纤维来确定通过分别使用ATP类似物(2-脱氧ATP)或具有改变的钙结合的TNC变体来调节交叉桥结合或细丝激活是否可以恢复人类成年DA骨骼肌的功能。

DESCRIPTION (provided by applicant): Arthrogryposis is a congenital contracture syndrome that affects 1 of 3000 live births. While the genetic causes of DA are in discovery, the biophysical mechanisms of how these mutations cause life-long contractures remains unknown. We have recently acquired and reported on contractile characterization for one DA patient, exhibiting an MYH3 R672C mutation. This mutation decreased steady state force production and increased relaxation time in single skinned myofibers from a human skeletal muscle biopsy, and our data suggest this may occur via altered crossbridge binding and cycling kinetics. The mechanism of how this occurs remains unknown. As such, there is much to be learned from contractile assessments from patient biopsies containing mutations in both thin and thick filament sarcomere proteins. I hypothesize that there are differences in how thin filament protein mutations and thick filament protein mutations result in altered contractures. Specifically, I expect thick filament mutations, such as MYH3 R672C and MYH8 R672H, to lead to direct changes in myosin (crossbridge) binding, detachment and interaction with actin. These mutations lead to a severe form of DA, Freeman-Sheldon Syndrome. In contrast, I expect thin filament mutations, such as TNNI2 R174Q andTNNT3 R63H, to lead to changes crossbridge binding via altered Ca2+ sensitivity and cooperativity of thin filament activation. These mutations lead to a slightly less severe form of DA, Sheldon-Hall syndrome. By determining functional changes and the causal mechanisms, we can begin to develop targeted therapies that would restore function on the molecular and cellular level. I plan to do so with three aims, focusing on muscle contractility of muscle fibers and muscle myofibrils. I will use muscle fibers to determine maximal force production, crossbridge binding, and Ca2+ sensitivity of force. I will use muscle myofibrils to determine thin filament activation and relaxation kinetics from single isolated myofibrils from human skeletal muscle biopsies. Finally, I will use muscle fibers and potentially myofibrils to determine if modulation of crossbridge binding or thin filament activation through use of an ATP analogue (2-deoxyATP) or TnC variants with altered Ca2+ binding, respectively, can restore function to human adult DA skeletal muscle.