Mechanisms of DUX4 mediated FSHD pathology

DUX4 介导的 FSHD 病理机制

• 批准号: 8506320
• 负责人: Peter L Jones
• 金额: $ 49.96万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-03 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8506320
• 关键词: 4q35; Adult; Affect; Biological Models; Biopsy; C-terminal; Candidate Disease Gene; Cell Death; Cell physiology; Cells; Child; Chromosomes; Clinical; Code; Consensus; Contracts; D4Z4; DNA; DNA Methylation; Development; Disease; Epigenetic Process; Facioscapulohumeral Muscular Dystrophy; Family; Gene Expression; Gene Targeting; Genes; Genetic; Goals; Individual; Laboratories; Lead; Length; Lesion; Libraries; Link; Mediating; Messenger RNA; Modeling; Molecular; Molecular Profiling; Muscle; Muscle Cells; Muscle Fibers; Muscle Weakness; Muscular Dystrophies; Mutation; Myopathy; Pathogenesis; Pathology; Pathway interactions; Patients; Phase; Poly A; Production; Protein Isoforms; Proteins; RNA Splicing; Regulation; Regulatory Element; Research; Research Design; Role; Staging; Testing; Therapeutic; Time; Variant; base; cohort; cytotoxic; histone modification; in vivo; mRNA Expression; novel therapeutics; overexpression; promoter; public health relevance; research study; telomere; therapy design

DESCRIPTION (provided by applicant): Our proposed experiments will identify mechanisms that cause mis-expression of DUX4 and lead to pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). FSHD is the most prevalent myopathy afflicting both children and adults, but no therapy is known. However, FSHD research has now entered an important new stage as DUX4 has emerged from studies in multiple laboratories, including our own, as the consensus FSHD candidate gene. Current evidence supports a model for FSHD pathology in which stable expression of a polyadenylated DUX4 mRNA splicing variant, termed DUX4-fl (fl = full-length), leads to production of a pathogenic DUX4-FL protein. This model is compatible with our finding, based on analysis of a large new library of myogenic cells and biopsies, that DUX4-fl mRNA and DUX4-FL protein are expressed at a much higher level and in a much higher fraction of myonuclei in FSHD than in healthy control muscle cells. Thus, in FSHD patients, clinically apparent muscle weakness may develop as DUX4-FL-induced pathological changes accumulate over time, whereas in healthy control muscles, the extremely low level of DUX4- FL is apparently insufficient to induce overt pathology. Although the FSHD-associated genetic lesion does not obviously alter protein coding or mRNA sequences, there is strong evidence that aberrant epigenetic regulation underlies DUX4 mis-expression. Our preliminary studies, for example, showed that DUX4-fl expression levels in FSHD myogenic cells could indeed be altered by manipulations of epigenetic status, e.g., DNA methlyation. Based on these epigenetic studies and our complementary analyses of DUX4-FL expression, we propose and will test the hypothesis that epigenetic dysregulation leads to aberrantly increased expression of DUX4-fl in FSHD vs. unaffected myogenic cells and thus to pathology. In Specific Aim 1, we wil identify epigenetic and non-epigenetic mechanisms that regulate DUX4-fl mRNA expression levels in skeletal muscle cells. In Specific Aim 2, we will analyze DUX4-FL expression in biopsies and also use single cell analyses to determine how DUX4-FL expression alters the fate of FSHD vs. unaffected cels. In Specific Aim 3, we will identify gene networks that are differentially regulate by the DUX4 protein isoforms and determine if they are disrupted in DUX4-fl-expressing FSHD cells. The results of our studies, in which we will systematically investigate the expression, regulation, and function of DUX4 isoforms in FSHD and control muscle cells, will provide the vital information needed to guide development of FSHD therapies specifically targeted to DUX4. !

描述（由申请人提供）：我们提出的实验将确定导致 DUX4 错误表达并导致面肩肱型肌营养不良症 (FSHD) 发病机制的机制。 FSHD 是儿童和成人中最常见的肌病，但尚无治疗方法。然而，FSHD 研究现已进入一个重要的新阶段，因为 DUX4 已从多个实验室（包括我们自己的实验室）的研究中脱颖而出，成为共识的 FSHD 候选基因。目前的证据支持 FSHD 病理学模型，其中多腺苷酸化 DUX4 mRNA 剪接变体（称为 DUX4-fl（fl = 全长））的稳定表达会导致致病性 DUX4-FL 蛋白的产生。该模型与我们的发现相一致，基于对大量新的生肌细胞和活组织检查文库的分析，与 FSHD 相比，DUX4-fl mRNA 和 DUX4-FL 蛋白的表达水平要高得多，并且在肌核中的比例要高得多。在健康的控制肌肉细胞中。因此，在 FSHD 患者中，随着 DUX4-FL 诱导的病理变化随着时间的推移累积，可能会出现临床上明显的肌肉无力，而在健康对照肌肉中，极低水平的 DUX4-FL 显然不足以诱导明显的病理。尽管 FSHD 相关的遗传损伤不会明显改变蛋白质编码或 mRNA 序列，但有强有力的证据表明，异常的表观遗传调控是 DUX4 错误表达的基础。例如，我们的初步研究表明，FSHD 肌原细胞中的 DUX4-fl 表达水平确实可以通过表观遗传状态（例如 DNA 甲基化）的操作来改变。基于这些表观遗传学研究和我们对 DUX4-FL 表达的补充分析，我们提出并将检验以下假设：与未受影响的肌原细胞相比，表观遗传失调导致 FSHD 中 DUX4-fl 表达异常增加，从而导致病理学。在具体目标 1 中，我们将确定调节骨骼肌细胞中 DUX4-fl mRNA 表达水平的表观遗传和非表观遗传机制。在具体目标 2 中，我们将分析活检中的 DUX4-FL 表达，并使用单细胞分析来确定 DUX4-FL 表达如何改变 FSHD 与未受影响细胞的命运。在具体目标 3 中，我们将鉴定受 DUX4 蛋白亚型差异调节的基因网络，并确定它们在表达 DUX4-fl 的 FSHD 细胞中是否被破坏。我们的研究结果将系统地研究 DUX4 同工型在 FSHD 和对照肌肉细胞中的表达、调节和功能，将为指导专门针对 DUX4 的 FSHD 疗法的开发提供所需的重要信息。 ！