Heterozygous MH knock-in mice model Human MH susceptibility

项目1的长期目标是确定兰尼定受体(RyR1)的作用机制 肌病、恶性高热和中枢性核心病(MH/CCD)。我们的近期目标是 在小鼠身上建立合适的人类疾病模型，并用它们来研究RyR1的突变是如何改变的 细胞内钙稳态，并建立一种非侵入性诊断试验，具有高度 特异性和敏感性。 假设一：杂合子MH“敲入”小鼠模型人类MH易感性。 A 1.1。创建一个热点区域2 MH‘敲入’小鼠品系(RyR1 G2434R)。除了我们最近 建立了R163C(区域1)和T4826I(区域3)RyR1MHS小鼠。A1.2确定以下各项之间的关系 肌萎缩侧索硬化症的临床表型和肌浆内静息游离钙([Ca~(2+)]_2) 对每只杂合子MH/CD小鼠进行体外实验。A1.3确定IVCT和 肌浆静息[Ca~(2+)]在人体活检标本的肌管中。A1.4以确定基因是否 背景可以挽救在纯合子MH/CD小鼠中看到的出生致命表型。A1.5以确定是否 遗传背景会降低或提高杂合子MH/CD小鼠的[Ca~(2+)]i或对氟烷的敏感性。 假说二：导致人类MH/CD基因突变增加被动RyR1“泄漏”和改变 通过加强幼儿保育和社会经济发展而实现的欧共体耦合的动力学。 A 2.1分析杂合子和纯合子的MH/CCD肌管在EC偶联和 钙离子进入的两种形式，ECCE和SOCE。A 2.2确定氟烷和丹曲林如何增强和 减少MH/CCD型肌管中异常的钙信号。A 2.3验证小鼠MH/CD肌管模型 从具有类似突变的人身上获得的肌管的结果。 假设III：“漏斗”方法可用于创建一种非侵入性筛查试验 MH既有高度的特异性，又有高度的敏感性。 A 3.1。我们将分析已知MHS RyR1突变患者的全血转录图谱 并使用漏斗方法选择一组预测性标记用于未来的MHS筛查。

The long-term goal of Project 1 is to define the mechanisms responsible for the ryanodine receptor (RyR1) myopathies, malignant hyperthermia and central core disease (MH/CCD). Our immediate objectives are to create suitable models for human disease in mice and use them to study how mutations of RyR1 alter intracellular Ca2+ homeostasis and to create a noninvasive diagnostic test for MH that has a high degree of specificity and sensitivity. HYPOTHESIS I: Heterozygous MH "knock-in" mice model Human MH susceptibility. A 1.1. To create a "hot-spot" region 2 MH 'knock in' mouse line (RyR1 G2434R). In addition to our recently created R163C (region 1) and T4826I (region 3) RyR1 MHS mice. A 1.2.To determine the relationship between the clinical MH phenotype and myoplasmic resting free calcium ([Ca2+],) in vivo, with contracture properties in vitro for each heterozygous MH/CCD mouse. A1.3 To determine the relationship between IVCT and myoplasmic resting [Ca2+] in myotubes from human biopsy samples. A1.4 To determine if genetic background can rescue the birth lethal phenotype seen in homozygous MH/CCD mice. A1.5 To determine if genetic background will lower or raise [Ca2+]i or sensitivity to halothane in heterozygous MH/CCD mice. HYPOTHESIS II: Mutations responsible for human MH/CCD increase passive RyR1 "leak" and alter the dynamics of EC coupling by enhancing ECCE and SOCE. A 2.1 To analyze heterozygous and homozygous MH/CCD myotubes for abnormalities in EC coupling and two forms of Ca2+ entry, ECCE and SOCE. A 2.2 To establish how halothane and dantrolene enhance and diminish aberrant Ca2+ signaling in MH/CCD myotubes. A 2.3 To validate murine MH/CCD myotube model results with myotubes obtained from humans with an analogous mutation. Hypothesis III: The "Funnel" approach can be used to create a non-invasive screening test for MH that has both a high degree of specificity and a high degree of sensitivity. A 3.1. We will analyze whole blood transcriptional profiles from patients with known MHS RyR1 mutations and use the Funnel approach to select a set of predictive markers to be used for future MHS screening.