Cardiac Functional and Structural Implications of Lamin A/C Mutations

 描述(申请人提供)：心肌病和心律失常是发病率高、治疗有限的疾病。尽管已发现大量基因与这些疾病的病因有关，但转化性研究--即实际应用遗传知识来改进对受影响个人及其家人的筛查、诊断和治疗--一直受到限制。一个主要的障碍是缺乏功能研究，以了解在多个生理尺度(细胞到组织)上的基因型和新出现的表型之间的关系，并确定导致家庭之间和家庭内临床变异的因素。这项拟议的研究集中在三个受影响的家庭中，每个家庭都有不同的LMNA基因突变。LMNA编码核层的主要蛋白质，核膜是与细胞核和细胞骨架相互作用的核膜结构基质。在这个提案中，我们将在多个长度尺度上检验LMNA突变与结构、组织和功能缺陷相关的假设。在家庭之间，缺陷的严重程度与LMNA突变的类型有关；在家庭内部，严重程度受到额外的遗传因素的影响。我们的长期目标是直接从患者那里建立体外疾病模型，以了解突变和遗传修饰物如何影响适当的细胞结构、组织结构和收缩功能。在具体目标1中，我们将使用外显子组测序和体外组织工程技术来评估LMNA患者和对照组成纤维细胞在细胞和核形态、细胞内结构、运动性以及组织自组装和结构方面的基因组变异和缺陷。在特定的目标2中，我们将从成纤维细胞中分离出诱导多能干细胞，并分析来自LMNA患者和对照组的诱导多能干细胞来源的心肌细胞的细胞结构和组织结构。在具体目标3中，我们将使用RNA测序来测试基因表达的改变，并使用“芯片上的心脏”来表征来自LMNA患者和对照组的iPS来源的心肌细胞的收缩功能(频率、收缩、舒张期和抽动应激)。这些结果结合遗传和表型变异的信息，将暗示我们将进一步研究的途径和功能。了解基因型和新出现的表型之间的复杂关系，并确定修饰因素，将有助于深入了解心脏病的发病机制，并有助于开发新的预防、诊断和治疗策略。

 DESCRIPTION (provided by applicant): Cardiomyopathies and arrhythmia are conditions with high morbidity and limited therapies. Although a vast number of genes have been discovered to contribute to the etiology of these diseases, translational research- the practical application of genetic knowledge to improve screening, diagnosis, and treatment for affected individuals and their families-has been limited. One major obstacle is the lack of functional studies to understand the relationship between genotype and emergent phenotype at multiple physiological scales (cells to tissues) and to identify factors that cause clinical variability between and within families. The proposed study focuses on three affected families each with different mutation in the Lamin A/C (LMNA) gene. LMNA encodes the main protein of the nuclear lamina, the structural matrix of the nuclear envelope that interacts with both the cell nucleus and cytoskeleton. In this proposal, we will test the hypothesis that LMNA mutations are associated with defects in the structure, organization, and function at multiple length-scales. Between families, the severity of the defects is associated with the type of LMNA mutation; and within families, severity is modified by additional genetic factors. Our long term goals are to develop in vitro disease models directly from patients to understand how proper cell structure, tissue organization, and contractile function are affected by the mutation and genetic modifiers. In Specific Aim 1, we will use exome sequencing and in vitro tissue engineering techniques to evaluate genomic variation and defects in cell and nuclear morphology, intracellular architecture, motility, and tissue self-assembly and architecture in fibroblasts from LMNA patients and controls. In Specific Aim 2, we will derive induced pluripotent stem cells (iPS) from fibroblasts and analyze cell structure and tissue organization of iPS-derived cardiomyocytes from LMNA patients and controls. In Specific Aim 3, we will use RNA sequencing to test for altered gene expression and the "Heart-on-a-Chip" to characterize contractility function (frequency, systolic, diastolic, and twitch stresses) of iPS-derived cardiomyocytes from LMNA patients and controls. These results combined with information of genetic and phenotypic variances will imply pathways and functionalities we will further study. Understanding of the complex relationship between genotype and emergent phenotype and identifying modifying factors will provide insight into the mechanism of heart disease and may assist in the development of new preventative, diagnostic, and therapeutic strategies.