Role of troponin T isoforms in nemaline myopathy

描述(由申请人提供)：阿米什线状肌病(ANM)是一种常染色体隐性遗传性肌肉疾病，发现于宾夕法尼亚州和俄亥俄州的旧秩序阿米什人中，每500名新生儿中就有1人受到影响。ANM等位基因包含慢速骨骼肌肌钙蛋白T(TNT)基因(TNNT1)的无义突变，导致TNT蛋白179位氨基酸截断。这种被截断的慢速TNT不能结合到肌原纤维中，并在肌细胞内降解。典型的ANM患者会出现肌肉震颤、痉挛和肌张力低下。受影响的婴儿在出生时临床上是正常的，但很快就会出现ANM症状，通常会导致第二年死于呼吸衰竭。目前还没有有效的治疗方法。本研究项目旨在了解ANM的发病机制和ANM肌肉的病理生理学，以期开发出治疗该疾病的最佳目标。提出了以下三个具体目标：目的I：建立慢TnT缺乏的动物模型。我们将研究慢TNT基因敲除小鼠的骨骼肌功能，在该模型中，慢TNT基因表达降低导致慢TNT蛋白减少，肌肉萎缩，并转向快速纤维表型，耐疲劳能力降低。我们还将研究ANM无义突变敲入慢速TNT基因的小鼠模型的表型，以探讨其发病机制和疾病进展。目的：研究慢TnT基因单倍体和截短型慢TnT在ANM发病机制和病理生理中的条件效应。ANM杂合子报告了周围肌肉症状。我们将研究一半剂量的慢TNT基因对杂合子慢TNT敲入突变小鼠肌肉功能的潜在影响。非肌丝相关的TNT片段具有细胞毒性。我们将研究ANM慢TNT片段的这种潜在的细胞毒性是否以及如何导致ANM患者的肌肉退化。目的：研究心肌TnT在骨骼肌中的调节和功能，为ANM患者的治疗代偿提供理论依据。我们在ANM的一个亚型中发现了证据，表明ANM中缓慢的骨骼TNT功能的丧失可能被骨骼肌中心肌TNT的持续表达所部分补偿。这种代偿性心脏TNT基因表达的功能意义和激活将在蛋白质、肌肉细胞和组织以及动物水平进行研究，以开发针对ANM的特定治疗方法。在另一种方法中，也将使用新出现的试剂来探索抑制ANM无义终止密码子。基于我们迄今取得的进展，这些研究采用了最先进的分子遗传学、生化、细胞生物学和生理学方法和新颖的实验系统。这一结果将大大推动对这种毁灭性疾病的有效治疗。公共卫生相关性：线虫性肌病是一组神经肌肉疾病，以肌肉无力和骨骼肌纤维中的杆状“线虫状”包涵体为特征。所有已知的遗传性线虫肌病都是由肌瘤细丝蛋白突变引起的。阿米什线状肌病(ANM)是一种致命性遗传性线状肌病，在宾夕法尼亚州和俄亥俄州的旧秩序阿米什社区中发病率非常高(每500名新生儿中就有1例)。ANM的遗传学基础是编码慢速骨骼肌肌钙蛋白T(TnT)亚型的基因的单核苷酸无义突变，TnT是一种肌肉特异性钙调节蛋白。这种无义突变截断了179位氨基酸的缓慢TNT多肽，使TNT蛋白无法结合到肌原纤维中，并被迅速降解。这种功能丧失的机制与该病的常染色体隐性遗传相一致。典型的ANM患者会出现肌肉震颤、痉挛和肌张力低下。症状在出生时很轻微，但很快就会恶化，通常会导致第二年死于呼吸衰竭。目前还没有有效的治疗方法。虽然ANM在普通人群中是一种罕见的疾病，但其一致破坏性的进展与已经阐明的分子原因相结合，值得进一步研究。在宾夕法尼亚州和俄亥俄州，每年有两到三个ANM婴儿出生；没有一个能活到3岁。我们目前是世界上唯一致力于ANM的研究小组，到目前为止，我们的进展已经给受影响的家庭和整个阿米什人社区带来了在不久的将来进行有效治疗的希望。这个研究项目旨在通过研究TNT亚型的功能和调节来进一步推动我们在治疗方面的进展。我们将研究慢性TNT缺乏的动物模型，以了解ANM的病理和肌肉功能。我们将研究减少慢TNT基因剂量对肌肉功能的潜在影响，以及截短的慢TNT与ANM肌肉退变的联系的细胞毒性。在探索抑制ANM无义终止密码子的可能性的同时，我们将重点研究慢性TNT缺陷骨骼肌中代偿性心肌TNT表达的功能意义和激活，以用于ANM的特异性治疗。ANM是唯一已知的由TNT基因隐性突变引起的疾病。通过研究ANM的病理生理学，我们还将对不同TNT亚型的功能意义以及横纹肌收缩的钙调节获得重要的见解，这是生物学和医学中的一个基本课题。

DESCRIPTION (provided by applicant): Amish Nemaline Myopathy (ANM) is an autosomal recessive muscle disorder found among the Old Order Amish in Pennsylvania and Ohio, affecting 1 out of every ~500 births. The ANM allele contains a nonsense mutation in the slow skeletal muscle troponin T (TnT) gene (TNNT1), which results in truncation of the TnT protein at amino acid 179. This truncated slow TnT cannot incorporate into myofibrils and is degraded inside the myocyte. Phenotypically, individuals with ANM suffer from muscle tremors, contractures and hypotonia. The affected infants are clinically normal at birth but rapidly develop the ANM symptoms that usually result in death from respiratory failure during the second year. No effective treatment is available. This research project aims to understanding the pathogenesis of ANM and the pathophysiology of ANM muscle for the optimal goal of developing a cure of the disease. The following three specific aims are proposed: Aim I: To characterize animal models deficient in slow TnT. We shall study the skeletal muscle function in a slow TnT knockdown mouse model in which lowered slow TnT gene expression results in decreased slow TnT protein, muscle atrophy and a switch to fast fiber phenotypes with decreased tolerance to fatigue. We shall also study the phenotype of a mouse model in which the ANM nonsense mutation is knocked in the slow TnT gene to investigate the pathogenesis and disease progression. Aim II: To examine the conditional effects of slow TnT gene haploidy and cytotoxicity of the truncated slow TnT on ANM pathogenesis and pathophysiology. ANM heterozygotes have reported circumstantial muscle symptoms. We shall investigate the potential effect of half dosage of slow TnT gene on muscle function in heterozygote slow TnT knock-in mutant mice. Non-myofilament-associated TnT fragments exhibit cytotoxicity. We shall examine whether and how this potential cytotoxicity of the ANM slow TnT fragment contributes to muscle degeneration in ANM patients. Aim III: To study the regulation and function of cardiac TnT in skeletal muscle in order to develop therapeutic compensation in ANM patients. We have found evidence in a subtype of ANM that the loss of slow skeletal TnT function in ANM may be partially compensated for by the continuing expression of cardiac TnT in skeletal muscles. Functional significance and activation of this compensatory cardiac TnT gene expression will be investigated at protein, muscle cell and tissue, and animal levels toward the development of a specific treatment for ANM. In an alternative approach, suppression of the ANM nonsense stop codon will also be explored using emerging reagents. Based on progresses we have made to date, these studies employ state-of-the-art molecular genetic, biochemical, cell biological and physiological methods and novel experimental systems. The results will significantly further the pursuit of an effective therapy for this devastating disease. PUBLIC HEALTH RELEVANCE: Nemaline myopathies are a group of neuromuscular disorders characterized by muscle weakness and rod-shaped "nemaline" inclusions in skeletal muscle fibers. All known inherited nemaline myopathies are caused by mutations in sarcomeric thin filament proteins. Amish Nemaline Myopathy (ANM) is a lethal inherited nemaline myopathy present at very high incidences (1 out of 500 births) in the Old Order Amish communities in Pennsylvania and Ohio. The genetic basis of ANM is a single nucleotide nonsense mutation in the gene encoding the slow skeletal muscle isoform of troponin T (TnT), a muscle-specific Ca2+- regulatory protein. This nonsense mutation truncates the slow TnT polypeptide at amino acid 179 and renders the TnT protein incapable of incorporating into myofibrils and being rapidly degraded. This loss of function mechanism is consistent with the autosomal recessive inheritance of the disease. Phenotypically, individuals with ANM suffer from muscle tremors, contractures and hypotonia. Symptoms are trivial at birth but rapidly worsen, usually resulting in death from respiratory failure during the second year. No effective treatment is currently available. Although ANM is a rare disease in the general population, its uniformly devastating progression combined with an already elucidated molecular cause merits much further investigation. Two to three ANM babies are born every year in Pennsylvania and Ohio; none will live to be 3 years old. We are currently the only research team in the world working on ANM and our progress thus far has already given the affected families and the general Amish community a hope for effective treatments in the near future. This research project aims to further our progress towards the development of a therapy by investigating TnT isoform function and regulation. We will study slow TnT-deficient animal models for the understanding of ANM pathology and muscle function. We will examine the potential effect of decreased slow TnT gene dosage on muscle function and the cytotoxicity of truncated slow TnT for links to muscle degeneration in ANM. Together with exploring the possibility of suppressing the ANM nonsense stop codon, we will focus on investigating the functional significance and activation of compensatory cardiac TnT expression in slow TnT-deficient skeletal muscle for use as a specific treatment for ANM. ANM is the only known disease caused by a recessive mutation in a TnT gene. By investigating the pathophysiology of ANM, we will also gain important insights into the functional significance of different TnT isoforms and the Ca2+-regulation of striated muscle contraction, a fundamental topic in biology and medicine.