The Role of mu-Crystallin in Facioscapulohumeral Muscular Dystrophy

描述（由申请人提供）：面肩肱骨肌营养不良症（FSHD）是一种常染色体显性疾病，遗传上与人类染色体位点4q35-qter相关。该区域与FSHD相关的遗传异常涉及4号染色体端粒上3.3 kb重复序列（D4Z4重复序列）的缺失。然而，这些重复序列的缺失并不足以引起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，并且还继承了4号染色体上4qA的4qA161单倍型。在重复区或顺式重复区的基因上调以及与4q35靠近核膜相关的因素已被提出作为机制，并得到一些实验支持，但基因阵列和定量RT-PCR研究未能证实这一假设。因此，可能由这些缺失引起的基因表达变化仍不清楚。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斑点，在我所研究的所有FSHD样本中都大量存在，但在健康和患病对照中却没有。等电点为5.08，分子质量为~34 kDa， 3个肽序列鉴定该位点为mu-crystallin，又称nadph依赖性甲状腺结合蛋白。与对照组相比，用单特异性抗体对mu-晶体蛋白的免疫印迹证实其在FSHD三角肌中存在高水平。mu- crystallin高度过表达的事实与FSHD的常染色体显性特征是一致的，这通常与“功能获得”突变有关。由于甲状腺激素（T3）在肌肉分化中起核心作用，mu-crystallin的过度表达可能部分通过改变T3活性参与FSHD的发病机制。值得注意的是，FSHD患者的内耳和视网膜都受到影响，而mu- crystallin的突变与耳聋和视网膜缺陷有关。在这里，我提出了3个具体目标，以严格检验mu-crystallin上调与FSHD特异性相关并致病的新假设：(I)检查来自年龄和性别匹配个体的不同肌肉的活组织检查，以及从这些样本中分离的不同蛋白质部分，以了解mu-crystallin在FSHD肌肉中的水平是否始终高于对照组；（ii）利用定量RT-PCR和序列分析了解mu-crystallin在FSHD患者肌肉中的转录是否上调，以及与健康和患病对照肌肉相比，FSHD患者肌肉中mu-crystallin转录本的序列是否始终存在变化。（iii）利用体内电穿孔和转基因小鼠过度表达不同水平的mu- crystallin来评估其在体内肌肉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，他们将对我们理解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.