Non-heritable genetic diseases of the skeletal system: Pathogenesis and Treatment

描述(申请人提供)：这项申请的重点是大量的儿童和成年人谁有影响骨骼的疾病是遗传的，但不是遗传的。这些疾病会导致严重的发病率。Gorham-Stout病(GSD)、全身性淋巴异常(GLA)、骨囊肿和肿瘤就是这些疾病的例子。这项应用的合作研究人员开发了一种方法，通过将从患者的受影响组织和未受影响的组织中提取的DNA或RNA的大规模并行测序与检测体细胞马赛克突变的计算算法相结合，从而确定这些类型疾病的遗传原因。这种方法不依赖于前瞻性地获得新鲜切除的皮损组织，因为它使用冰冻组织和存档的福尔马林固定石蜡包埋的病理样本。我们通过使用我们的方法来验证我们的方法，发现PIK3CA中的体细胞马赛克激活突变导致丁香综合症。我们现在建议将我们的工作扩展到其他遗传性、非遗传性疾病。我们已经确定了几个特征良好的患有GSD、GLA和骨肿瘤等疾病的患者队列，我们一直在从这些人身上收集受影响和未受影响的组织样本。我们目前收集的样本包括6例GLA，6例GSD，20例骨巨细胞瘤，38例软骨母细胞瘤，8例软骨粘液样纤维瘤的皮损组织。在这项提议中的一个目标是，我们将从受影响的组织和未受影响的组织中准备条形码DNA和cDNA文库，执行大规模并行测序，并使用已经使我们能够识别其他疾病的致病突变的计算算法。我们希望成功地识别这些新疾病中的许多致病突变。目的我们的建议中的两个建立在我们的发现之上，即当PIK3CA激活突变以体细胞镶嵌形式存在时，会导致图案缺陷、畸形和过度生长。我们有一只小鼠可以有条件地激活p.1047H&gt；R突变，我们已经在丁香、KTS、FIL和孤立的淋巴畸形患者中发现了这种突变；我们很快就会有一只小鼠，我们可以在其中有条件地激活然后灭活另一种致病的PIK3CA突变，p.420C&gt；R。我们将使用这些小鼠来了解PIK3CA相关疾病中发生的畸形和过度生长的病因，并确定这些问题是否可以预防、延迟或逆转。完成这些目标将把基因与以前无法通过基因分析的疾病患者的表型联系起来，对于PIK3CA相关表型的患者，将确定病变是如何形成的，以及抑制PIK3CA是否是一种可行的治疗策略。

DESCRIPTION (provided by applicant): This application focuses on the large number of children and adults who have diseases affecting the skeleton that are genetic, but not heritable. These disorders cause significant morbidity. Gorham-Stout Disease (GSD), Generalized Lymphatic Anomaly (GLA), bone cysts and tumors exemplify these diseases. The co-investigators of this application have developed an approach for identifying genetic causes for these types of disorders by combining massively parallel sequencing of DNA or RNA - recovered from a patient's affected tissues and unaffected tissues - with computational algorithms that detect somatic mosaic mutations that are either solely in sequence from affected tissue or are enriched in sequence from affected tissue compared to unaffected tissue. This approach does not rely on prospectively obtaining freshly excised lesional tissue, since it works using frozen tissue and archived formalin fixed paraffin embedded pathologic samples. We validated our approach by employing it to discover that somatic mosaic activating mutations in PIK3CA are responsible for CLOVES syndrome. We now propose to extend our work to other genetic, non-hereditary conditions. We have ascertained several well-characterized patient cohorts affected with diseases such as GSD, GLA, and bone tumors, and we have been collecting affected and unaffected tissue samples from these individuals. Our sample collection currently includes lesional tissue from 6 patients with GLA, 6 patients with GSD, 20 patients with giant cell tumors, 38 patients with chondroblastoma, 8 patients with chondromyxoid fibroma. In Aim One of this proposal, we will prepare bar-coded DNA and cDNA libraries from affected tissue and unaffected tissue, perform massively parallel sequencing, and use computational algorithms that have already enabled us to identify causative mutations in other disorders. We expect to successfully identify causative mutations in many of these new disorders. Aim Two of our proposal builds upon our discovery that PIK3CA activating mutations cause patterning defects, malformations, and overgrowths when present in somatic mosaic form. We have a mouse in which we can conditionally activate the p.1047H>R mutation, which we have found in persons with CLOVES, KTS, FIL, and isolated lymphatic malformations, and we will soon have a mouse in which we can conditionally activate and then inactivate a different disease-causing Pik3ca mutation, p.420C>R. We will use these mice to understand the pathogeneses of malformations and overgrowths that occur in PIK3CA-associated disorders and to determine whether these problems can be prevented, delayed, or reversed. Completion of these aims will link genes to phenotypes in patients whose diseases were previously impenetrable to genetic analyses and, for patients with PIK3CA-associated phenotypes, determine how lesions form and whether PIK3CA inhibition is a viable treatment strategy.