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

描述（由申请人提供）：此应用程序主要针对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。这些疾病引起严重的发病率。Gorham-Stout病（GSD）、广泛性淋巴异常（GLA）、骨囊肿和肿瘤是这些疾病的典型代表。该应用程序的共同研究人员已经开发出一种方法，通过结合从患者的受影响组织和未受影响组织中恢复的DNA或RNA的大规模平行测序，以及检测体细胞马赛克突变的计算算法，来识别这些类型疾病的遗传原因，这些突变要么仅在受影响组织的序列中，要么在受影响组织的序列中与未受影响组织相比富集。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石蜡包埋病理样本。我们通过使用它来验证我们的方法，发现PIK3CA的体细胞镶嵌激活突变是CLOVES综合征的原因。我们现在建议将我们的工作扩展到其他遗传的、非遗传的条件。我们已经确定了几个具有良好特征的患者队列，这些患者患有GSD、GLA和骨肿瘤等疾病，我们一直在收集这些个体的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患者、6例GSD患者、20例巨细胞瘤患者、38例成软骨细胞瘤患者、8例软骨粘液样纤维瘤患者的病变组织。在本提案的第一个目标中，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，进行大规模平行测序，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。我们期望在许多这些新疾病中成功地确定致病突变。我们的建议建立在我们的发现，PIK3CA激活突变导致图案缺陷，畸形，和过度生长时，存在于体细胞镶嵌形式。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和孤立淋巴畸形的人身上发现了这种突变，我们很快就会有一只小鼠，我们可以有条件地激活然后灭活另一种致病的Pik3ca突变p.420C>R。我们将使用这些小鼠来了解在pik3ca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以前无法通过遗传分析的疾病患者的表型联系起来，并且对于具有PIK3CA相关表型的患者，确定病变如何形成以及PIK3CA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.