Modeling Inheritable Myofibrillar Myopathy with Patient-Specific iPS Cells

 描述（由申请方提供）：在文献中发现了编码aB-晶状体蛋白的基因中的多个突变，这些突变导致骨骼肌病。α B-晶体蛋白是一种小分子量的热休克蛋白，已被证明是结蛋白、肌联蛋白和肌动蛋白的伴侣。此外，α B-晶状体蛋白通过对细胞周期退出和肌肉特异性调节转录因子MyoD的影响来影响骨骼肌分化。 一名携带CRYAB纯合隐性无义突变343 delT的患者在正常发育后，在四个月左右出现严重肌肉僵硬和变性的症状。患者的肌肉活检显示343 delT的致密、不规则染色，可能使人联想到与其它突变形式的aB-晶状体蛋白（如R120 G）一起可见的聚集体，其已显示也诱导中间丝蛋白结蛋白的聚集，从而促成疾病病理。患者的症状也与HSPAB/HSPB 2双敲除小鼠相似，尽管小鼠似乎具有较不严重和较晚发作的肌肉退化。因此，关于这种疾病病理学是由α B-晶状体蛋白功能丧失还是343 delT α B-晶状体蛋白毒性功能获得引起的问题仍然存在，以及这种影响是否在肌源性祖细胞、分化的肌管或两者中观察到。该提案旨在使用343 delT患者特异性诱导多能干细胞（iPSC）衍生的肌源性祖细胞和骨骼肌管来研究这个问题。iPSC在体外模拟细胞自主性疾病中提供了很大的实用性，因为它们具有持续的自我更新能力和多能分化潜力;它们也适合遗传操作。iPSC将通过现有方案分化为肌源性祖细胞和骨骼肌管。[对照iPSC已经通过在343 delT患者iPSC中的基因校正产生]，并且将用于表型比较以检查分化的肌管的结构和功能，以及分析在肌原祖细胞分化为肌管的整个过程中的细胞周期进展和基因表达。此外，已经从343 delT患者iPSC产生了无CD 40 AB的iPSC用于比较，以确定表型是否是由于343 delT突变的功能效应的丧失或获得。这些目标的成功完成将提供343 delT诱导的肌病的人类模型系统，并更好地理解突变体的表达和/或表达的缺乏如何促成肌病，以及伴侣蛋白aB-晶状体蛋白在人类骨骼肌分化和维持中的重要性。

 DESCRIPTION (provided by applicant): Multiple mutations in the gene encoding aB-crystallin, CRYAB, have been identified in the literatures that result in skeletal myopathy. αB-Crystallin is small molecular-weight heat shock protein that has been shown to function as a chaperone for desmin, titin, and actin. Additionally, αB-crystallin impacts skeletal muscle differentiation throgh exhibiting effects on cell cycle exit and a muscle-specific, regulatory transcription factor, MyoD. A patient harboring the homozygous recessive, nonsense mutation, 343delT, in CRYAB develops symptoms of severe muscle stiffness and degeneration around age four months, following normal development. The patient's muscle biopsy shows dense, irregular staining of 343delT, possibly reminiscent of the aggregates visible with other mutant forms of aB-crystallin, such as R120G, which has been shown to also induce the aggregation of an intermediate filament protein, desmin, contributing to the disease pathology. The patient's symptoms also bear similarities to the CRYAB/HSPB2 double knockout mouse, though the mouse seems to have less severe and later onset muscle deterioration. Therefore, the question as to whether this disease pathology results from loss of αB-crystallin function or gain of toxic function of 343delT αB-crystallin remains and whether this impact is seen in myogenic progenitor cells, differentiated myotubes, or both. This proposal aims to examine this question using 343delT patient-specific induced pluripotent stem cell- (iPSC) derived myogenic progenitors and skeletal myotubes. iPSCs offer great utility in modeling cell autonomous diseases in vitro because of their continuous ability to self-renew and pluripotent differentiation potential; they are also amenable to genetic manipulation. iPSCs will be differentiated to myogenic progenitors and skeletal myotubes by an existing protocol. [Control iPSCs have been generated by gene correction in 343delT patient iPSCs] and will be used for phenotypic comparisons to examine the structure and function of differentiated myotubes as well as analyze cell cycle progression and gene expression throughout differentiation of myogenic progenitors to myotubes. Additionally, CRYAB null iPSCs have been generated from 343delT patient iPSCs for comparison to determine if phenotypes are due to loss or gain of function effects of the 343delT mutation. Successful completion of these aims will provide a human model system of 343delT-induced myopathy and a better understanding of how expression of the mutant and/or lack of expression contribute to myopathy, as well as the importance of a chaperone, aB-crystallin, in human skeletal muscle differentiation and maintenance.