The HH: A Large Cohort of Patients with Congenital Myopathies of Uncertain Etiology

项目摘要 细胞内钙信号在肌肉中达到很高的强度，在那里它们是“兴奋- 收缩耦合“(ECC)过程。在横纹肌中，它们是由超分子组装产生的 我们将这种偶联命名为肌浆通道，它的关键是包括兰尼定受体(RyRs)。 网状结构(SR)。多种疾病由ECC异常引起；其中，聚集性恶性 体温过高是通过“CHCT”来诊断的，这是一种用咖啡因和氟烷进行的传统挑战。在一辆72- 患者样本中，我们发现大约20%的检测呈阳性，40%为阴性，40%为检测 模棱两可的意思是，他们对氟烷有过敏反应，但对咖啡因没有反应。临床工作发现，这些 病人，我们称之为“HH”，生病，肌肉疼痛，虚弱，对压力高度敏感，或 受热，或他汀类药物，并经历横纹肌溶解和其他挫折。这与大多数MH- 阳性患者，有众所周知的诱因易感性，但其他没有活动性疾病表型。 在这里，两个生理实验室与研究最多先天性非先天性心脏病的诊所合作 半球营养不良肌病(MHIU)提出一项约300人的综合研究 病人。每个接受测试的患者的详细临床和基因图像将通过以下方式匹配：(1)细胞水平 钙处理的量化(通过测量胞质中稳定和刺激的钙离子浓度和 在成体细胞和培养细胞中，以及这些隔室之间的稳定和刺激的通量 来自患者的活组织检查)，以及(2)来自单个SR功能测量的匹配的分子描述 CA释放患者来源的RyR1通道。这些测量中的许多将是在 人类细胞。研究结果将从“致病途径”的角度进行解释，这种途径追踪因果关系， 从主要缺陷(例如，RyR打开的过度倾向)开始解释和预测 发生在下游的多个变化。然后，这种机械知识将被用来设计治疗方法 干预措施，合理定制，以抵消主要缺陷或已建立的致病因素的主要驱动因素 小路。这可能包括细胞外介质离子组成的稳定变化，这是经典药物 丹曲林和/或一大套新合成的RyR抑制药物卡维地洛衍生物的应用 从母体药物改装而来，以消除其β-阻断作用。在新的衍生品中，有34种是 在RyR表达系统中进行了有利的预筛选。其中最好的，根据亲和力、有效性和 RyR-异构体特异性，将应用于单个人RyR1通道、肌管和肌纤维；他们的 结果将与丹曲林进行比较，并在#年建立的机制背景下进行解释 这个项目。我们的研究与临床密切相关，这使得我们有可能量身定做潜在的 治疗干预措施(最初由HH和MH队列的集体属性提供信息) 个体患者的表型(分子、细胞或器官)。在未来的迭代中，顶级治疗药物 范例将加入已经在MHIU进行的临床测试。(11/02/16修订版)

Project Summary Intracellular Ca signals reach great intensity in muscle, where they are key to the “Excitation- Contraction Coupling” (ECC) process. In striated muscles they are produced by a supramolecular assembly that we named the couplon, which crucially includes ryanodine receptors (RyRs), channels of the sarcoplasmic reticulum (SR). Multiple diseases arise from abnormal ECC; among them, the paradigmatic Malignant Hyperthermia is diagnosed by the “CHCT”, a conventional challenge with caffeine and halothane. In a 72- patient sample, we have found that roughly 20% tested positive, 40% were negative and 40% tested equivocally, meaning that they Hyper-reacted to Halothane, but not to caffeine. Clinical work found that these patients, which we call the “HH”, are sick, suffering from muscle pain, weakness, high sensitivity to stress, or heat, or statins, and experience rhabdomyolysis and other setbacks. This is in stark contrast with most MH- positive patients, who have a susceptibility to well-known triggers, but otherwise no active disease phenotype. Here, two physiology labs have teamed with the clinic that studies the greatest number of congenital non- dystrophic myopathies in the hemisphere (the MHIU) to propose a comprehensive study of approximately 300 patients. A detailed clinical and genetic picture of each tested patient will be matched by: (1) a cell-level quantification of Ca handling (from measurements of steady and stimulated Ca ion concentration in cytosol and SR, as well as steady and stimulated fluxes between these compartments in adult and cultured cells derived from patients’ biopsies), and (2) a matching molecular description, from measurements of function of single SR Ca release RyR1 channels derived from the patients. Many of these measurements will be the first done in human cells. The results will be interpreted in terms of “pathogenic pathways”, which track the causal chain, starting from a primary defect (e.g. an excessive tendency for RyR to open) to account for and predict the multiple changes that occur downstream. This mechanistic knowledge will then be used to devise therapeutic interventions, tailored rationally to offset the primary defect or the main drivers of the established pathogenic pathways. These may include steady changes in ion composition of the extracellular medium, the classic drug dantrolene and/or application of a large set of newly synthesized RyR-inhibiting drugs, carvedilol derivatives modified from the parent drug to eliminate its beta-blocking action. Among the novel derivatives, 34 were prescreened favorably in a RyR expression system. The best of these, identified based on affinity, efficacy and RyR-isoform specificity, will be applied to single human RyR1 channels, myotubes and myofibers; their outcomes will be compared to those of dantrolene and interpreted within the mechanistic context established in this project. The close bench-clinical correlation of our study makes it possible to tailor the design of potential therapeutic interventions (initially informed by the collective properties of the HH and MH cohorts) to the phenotype (molecular, cellular, or organismal) of individual patients. In future iterations, the top therapeutic paradigms will join clinical testing already going on at the MHIU. (Rev. 11/02/16)