The Role of mu-Crystallin in Facioscapulohumeral Muscular Dystrophy

描述(申请人提供)：面肩肩周型肌营养不良症(FSHD)是一种常染色体显性遗传病，与人类染色体4q35-QTER基因连锁。与FSHD相关的该区域的遗传异常涉及4号染色体端粒上3.3kb的重复序列(D4Z4重复)的缺失。然而，这些重复序列的缺失不足以导致FSHD。我们目前的理解是，据报道，所有有症状的FSHD患者的D4Z4重复单位少于10个，并且还遗传了4号染色体上4qA的4qA161单倍型。重复区域内或顺式基因上调以及与4q35接近核膜相关的因素被认为是机制，并得到了一些实验支持，但基因阵列和定量RT-PCR研究未能证实这一假说。因此，这些缺失可能导致的基因表达变化尚不清楚。我采用了蛋白质组学和细胞生物学相结合的方法来研究FSHD肌肉。我的初步结果表明，三角肌的可溶性部分显示出一个单一的蛋白质点，在我研究过的所有FSHD样本中都存在大量的这种蛋白质点，但在健康和疾病对照组中没有。等电点为5.08，分子质量约为34 kDa，3个多肽序列确定该斑点为结晶素，也称为NADPH依赖的甲状腺结合蛋白。免疫印迹证实，与对照组相比，Mu-晶体蛋白在FSHD三角肌中的水平较高。Mu-晶体蛋白高度过度表达的事实与FSHD的常染色体显性特征一致，这通常与“功能获得”突变有关。由于甲状腺激素(T3)在肌肉分化中起中心作用，Mu-晶体蛋白的过度表达可能部分通过改变T3活性而参与FSHD的发病。值得注意的是，FSHD患者的内耳和视网膜都会受到影响，u-晶体蛋白的突变与耳聋和视网膜缺陷有关。在这里，我提出了三个特定的目标来关键地检验这一新的假设，即Mu-crystallin的上调与FSHD特异地关联并导致FSHD：(I)检查来自年龄和性别匹配的不同肌肉的活组织标本，以及从这些样本中分离的不同蛋白质组分，以了解Mu-crystallin是否持续在FSHD肌肉中持续存在于与对照组相比的较高水平；(Ii)使用定量RT-PCR和序列分析来了解FSHD患者的肌肉中Mu-crystallin是否转录上调，以及与健康和疾病对照肌肉相比，FSHD患者肌肉中是否存在Mu-crystallin转录的序列变异。(Iii)使用体内电穿孔和过量表达不同水平的u-晶体蛋白的转基因小鼠来评估其在体内肌肉中过度表达的病理后果。由于我的新假设挑战了现有的范式，我的实验本质上是高风险的。如果成功，它们将对我们理解FSHD的分子机制产生重大影响。 公共卫生相关性：这项建议中的实验旨在测试新的假设，即u-晶体蛋白(CRYM)的表达增加足以导致骨骼肌病理。这项工作的动力来自于我的初步发现，与炎症性肌病和其他肌肉营养不良患者的健康人肌肉或肌肉相比，第三种最常见的肌肉营养不良-面肩肩周肌营养不良(FSHD)患者的肌肉中u-晶体蛋白的水平异常高。我的实验将确认u-晶体蛋白的表达增加是FSHD患者肌肉所特有的，它的上调是否发生在转录水平，以及该蛋白的长期过度表达是否会导致其他正常小鼠的肌肉营养不良。由于我的新假设挑战了现有的范式，我的实验本质上是高风险的。如果成功，它们将对我们理解FSHD的分子机制产生重大影响。

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease genetically linked to human chromosomal locus 4q35-qter. The genetic abnormality in this region associated with FSHD involves deletions of 3.3 kb repeating sequences (D4Z4 repeats) at the telomere of chromosome 4. However, deletions of these repeats are not sufficient to cause FSHD. Our current understanding is that all symptomatic FSHD patients reportedly have fewer than 10 D4Z4 repeating units and also have inherited the 4qA161 haplotype of 4qA on chromosome 4. Up-regulation of genes either within or in cis to the repeat region and factors associated with the proximity of 4q35 to the nuclear envelope have been proposed as mechanisms, with some experimental support, but gene array and quantitative RT-PCR studies have failed confirm this hypothesis. Thus, the changes in gene expression presumably caused by these deletions remain unclear. I have adopted a combination of proteomic and cell biological approaches to study FSHD muscle. My preliminary results indicate that the soluble fraction of deltoid muscles shows a single protein spot that is present in high amounts in all FSHD samples I have studied, but absent in healthy and diseased controls. The isoelectric point of 5.08, molecular mass of ~34 kDa, and 3 peptide sequences identified this spot as mu-crystallin, also known as NADPH-dependent thyroid binding protein. Immunoblots with mono-specific antibodies to mu-crystallin confirmed its presence at high levels in FSHD deltoid muscles compared to controls. The fact that mu- crystallin is highly over-expressed is consistent with FSHD's autosomal dominant character, which is normally associated with a "gain of function" mutation. As thyroid hormone (T3) plays a central role in the differentiation of muscle, the over-expression of mu-crystallin could contribute to pathogenesis in FSHD in part by altering T3 activity. Remarkably, the inner ear and the retina are both affected in FSHD patients, and mutations in mu- crystallin have been linked to deafness and to retinal defects. Here I propose 3 specific aims to test critically the novel hypothesis that up-regulation of mu-crystallin is specifically linked to, and pathogenic for, FSHD: (i) to examine biopsies from different muscles from age- and sex-matched individuals, and different protein fractions isolated from these samples, to learn if mu-crystallin is consistently present at higher levels in FSHD muscle compared to controls; (ii) to use quantitative RT-PCR and sequence analysis to learn whether mu- crystallin is transcriptionally upregulated in muscles from patients with FSHD and whether there are sequence variations in mu-crystallin transcripts consistently present in FSHD patients, compared to healthy and diseased control muscles. (iii) to use in vivo electroporation and transgenic mice over-expressing varying levels of mu- crystallin to assess the pathological consequences of its over-expression in muscle in vivo. As my new hypothesis challenges existing paradigms, my experiments are high risk in nature. If successful, they will have a significant impact on our understanding of the molecular mechanisms underlying FSHD. PUBLIC HEALTH RELEVANCE: The experiments in this proposal are designed to test the novel hypothesis that an increase in the expression of mu-crystallin (CRYM) is sufficient to cause pathology in skeletal muscle. The impetus for this work stems from my preliminary finding that mu-crystallin is present at abnormally high levels in muscles from patients with the third most common form of muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), as compared to healthy human muscle or muscle from patients with inflammatory myopathies and other muscular dystrophies. My experiments will confirm that the increased expression of mu-crystallin is specific to muscles from FSHD patients, whether its up-regulation occurs at the transcriptional level, and whether chronic over-expression of this protein can cause muscular dystrophy in otherwise normal mice. As my new hypothesis challenges existing paradigms, my experiments are high risk in nature. If successful, they will have a significant impact on our understanding of the molecular mechanisms underlying FSHD.