Identifying the Gene for Scleroderma in Tsk/2 mice that model Human SSc Subsets

描述（由申请人提供）：系统性硬化症（SSc）是一种复杂的炎性纤维化疾病，具有几种不同的临床表现。它可能有危及生命的并发症，而且没有治愈的方法。SSc患者表现出疾病严重程度的连续性，通常范围从局限于四肢的不太严重的纤维化到更严重且通常致命的弥漫性疾病。Tsk 2/+小鼠是SSc的模型。在我们的实验室中，Tsk 2突变已经被培育到同质近交系（C57 B1/6或B6）背景上，但是Tsk 2/+的基因是未知的，并且对其疾病的分子基础了解甚少。B6.Tsk2/+小鼠具有人类疾病的许多特征，包括皮肤紧绷、失调的细胞外基质沉积和显著的自身免疫。我们发现Tsk 2介导的自身免疫和纤维化体征随着年龄的增长而逐渐发展。SSc根据以下类型分为亚型： 对疾病症状的局限性和扩散性的影响。通过我们的合作者团队，我们现在 显示Tsk 2/+小鼠在4周时与“弥漫性-增殖性”人类疾病亚型的基因表达具有显著重叠。TGF 21在SSc中的疾病促进作用是众所周知的，并且我们发现来自Tsk 2/+小鼠的皮肤样品在TGF 21的下游靶点中有实质性增加。 在这里，我们建议确定Tsk 2基因，并了解其作用机制。这只小鼠提供了一个独特的机会来检查从出生起导致纤维化疾病多个临床参数的途径。我们的主要假设是，Tsk 2/+小鼠准确地代表了皮肤显示TGF β通路活化的弥漫性增殖性SSc患者的亚组，并且鉴定Tsk 2基因将提供对该组患者中疾病机制的独特见解。我们将从这些小鼠中分离纯合的Tsk 2突变体和野生型胚胎成纤维细胞，用于对两个等位基因间隔进行深度测序以找到基因。 我们将在全球基因谱研究中研究RNA表达，并使用相同的样本，我们将检查ECM的组织学证据。 失调我们将研究这种特性是否是细胞自主的，或者它是否需要协调一致的努力， 不同的细胞亚型，特别是树突细胞或肥大细胞，以及受影响区域的成纤维细胞和角质形成细胞。一旦我们确定了Tsk 2基因的身份，我们将在体外测试暴露于来自Tsk 2/+细胞的ECM组分后纤维化细胞行为的显著变化。来自三个不同机构的四位合作者26将共同解决有关SSc和我们的B6.Tsk2/+模型的关键问题：Col 3a 1是Tsk 2/+性状的潜在基因，还是突变发生在连锁位点？Tsk 2/+模型中的主要激发事件是什么？Tsk 2等位基因如何引发疾病？TGF-21何时以及如何发出ECM失调信号？ TGF-21信号传导的直接靶点是什么？ B6.Tsk2/+小鼠能否作为弥漫增殖型SSc的良好模型？ 公共卫生相关性：在Tsk 2/+小鼠中突变的特定基因尚不清楚，但这种突变的结果产生了一种与称为弥漫性增殖亚群的硬皮病类型非常相似的疾病。如果我们在基因发现、基因表达和细胞亚群研究方面取得成功，我们预计将发现几个治疗这种毁灭性疾病的“可药物化”靶点。至少，Tsk 2/+小鼠可能成为比其他现有模型更好的硬皮病临床前模型。

DESCRIPTION (provided by applicant): Systemic sclerosis (SSc) is a complex inflammatory fibrotic disease, with several distinct clinical manifestations. It can have life-threatening complications and there is no cure. SSc patients show a continuum of disease severity, generally ranging from less severe fibrosis limited to the extremities, to a more severe and often fatal diffuse disease. Tsk2/+ mice are a model for SSc. The Tsk2 mutation has been bred onto a homogeneous inbred (C57Bl/6, or B6) background in our laboratory, but the gene for Tsk2/+ is unknown, and little is understood about the molecular basis for their disease. B6.Tsk2/+ mice have many features of the human disease, including tight skin, dysregulated extracellular matrix deposition, and significant autoimmunity. We have found that Tsk2- mediated autoimmune and fibrotic signs develop progressively with age. SSc is classified into subtypes based on the limited vs. disseminated appearance of disease symptoms. With our team of collaborators, we now show that the Tsk2/+ mice at four weeks have a significant overlap in gene expression with the "diffuse- proliferative" human disease sub-type. The disease-promoting role of TGF21 in SSc is well-known, and we find that skin samples from Tsk2/+ mice have substantial increases in downstream targets of TGF21. Herein we propose to identify the Tsk2 gene and understand its mechanism of action. This mouse affords a unique opportunity to examine the pathways leading to the multiple clinical parameters of fibrotic disease from birth onward. Our lead hypothesis is that the Tsk2/+ mouse accurately represents the subset of diffuse- proliferative SSc patients whose skin shows activation of the TGFb pathway, and that identifying the Tsk2 gene will provide unique insight into the mechanism of disease in this group of patients. We will isolate homozygous Tsk2 mutant and wild type embryonic fibroblasts from these mice for purposes of deep sequencing the two allelic intervals to find the gene. We will study both RNA expression in global gene profiling studies and with the same samples, we will examine the resulting histological evidence of ECM dysregulation. We will study whether the trait is cell-autonomous or whether it requires the concerted efforts of different cell subtypes, especially dendritic cells or mast cells, and the fibroblasts and keratinocytes that are in the affected areas. Once we have the identity of the Tsk2 gene, we will test for significant changes in fibrotic cell behavior after exposure to ECM components from the Tsk2/+ cells in vitro. Together, the four collaborators 26 from three different institutions will address key questions about SSc and our B6.Tsk2/+ model: Is Col3a1 the gene underlying the Tsk2/+ traits or has the mutation occurred in a linked locus? What is the major inciting event in the Tsk2/+ model and how does the Tsk2 allele initiate disease? When and how does TGF21 signal ECM dysregulation? What is the direct target of TGF21 signaling? Can B6.Tsk2/+ mice be used as a good model for diffuse-proliferative SSc? PUBLIC HEALTH RELEVANCE: The specific gene that is mutated in the Tsk2/+ mouse is not known, but the result of this mutation creates a disease that closely resembles a type of Scleroderma called the Diffuse-proliferative subset. If we are successful in our gene discovery, gene expression and cell subset investigations, we anticipate that several 'druggable' targets for this devastating disease will be found. At the very least, the Tsk2/+ mouse may become a better alternative as a pre-clinical model of scleroderma than other existing models.