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

描述（由申请人提供）：本申请侧重于大量患有影响骨骼的遗传性但非遗传性疾病的儿童和成人。这些疾病导致显著的发病率。Gorham-Stout病（GSD）、全身性淋巴异常（GLA）、骨囊肿和肿瘤属于这些疾病。 本申请的共同研究者已经开发了一种用于鉴定这些类型的病症的遗传原因的方法，其通过将从患者的受影响组织和未受影响组织回收的DNA或RNA的大规模平行测序与检测体细胞嵌合突变的计算算法相结合来进行，所述体细胞嵌合突变或者仅在来自受影响组织的序列中，或者与未受影响组织相比在来自受影响组织的序列中富集。这种方法不依赖于前瞻性地获得新鲜切除的病变组织，因为它使用冷冻组织和存档的福尔马林固定的石蜡包埋的病理样品。我们通过使用它来发现PIK 3CA中的体细胞嵌合激活突变是CLOVES综合征的原因，从而验证了我们的方法。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受GSD，GLA和骨肿瘤等疾病影响的良好特征的患者队列，并且我们一直在从这些个体中收集受影响和未受影响的组织样本。我们的样本收集目前包括6例GLA患者，6例GSD患者，20例巨细胞瘤患者，38例软骨母细胞瘤患者，8例软骨粘液样纤维瘤患者的病变组织。在该提案的目标之一，我们将从受影响的组织和未受影响的组织中制备条形码DNA和cDNA文库，进行大规模并行测序，并使用已经使我们能够识别其他疾病中的致病突变的计算算法。我们希望能够成功地确定许多这些新疾病的致病突变。 我们的两个提议建立在我们的发现基础上，即PIK 3CA激活突变在以体细胞嵌合体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，我们可以有条件地激活p.1047H>R突变，我们已经在患有CLOVES，KTS，FIL和孤立的淋巴管畸形的人中发现了这种突变，我们很快就会有一只小鼠，我们可以有条件地激活然后消除一种不同的致病Pik 3ca突变，p.420C>R。我们将使用这些小鼠来了解PIK 3CA相关疾病中发生的畸形和过度生长的发病机制，并确定这些问题是否可以预防，延迟或逆转。 这些目标的完成将把基因与患者的表型联系起来，这些患者的疾病以前无法通过遗传分析，对于PIK 3CA相关表型的患者，确定病变如何形成以及PIK 3CA抑制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.