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

骨骼系统非遗传性遗传疾病：发病机制和治疗

• 批准号: 9245631
• 负责人: Matthew L Warman
• 金额: $ 38.94万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-03 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245631
• 关键词: Ablation; Adult; Affect; Alleles; Animal Model; Animals; Archives; Bar Codes; Benign; Biology; Biopsy; Bone Cysts; Bone neoplasms; Caring; Cells; Child; Chondroblastoma; Chondromyxoid Fibroma; Clinical Trials; Complex; Computational algorithm; DNA; DNA Library; DNA sequencing; Defect; Development; Diagnosis; Disease; Disease Progression; Enterobacteria phage P1 Cre recombinase; Evaluation; Face; Finding by Cause; Formalin; Freezing; Genes; Genetic; Genetic Counseling; Giant Cell Tumors; Glean; Goals; Gorham-Stout disease; Hereditary Disease; Human; Individual; Intervention; Klippel-Trenaunay-Weber Syndrome; Knock-in; Learning; Lesion; Link; Lipomatosis; Lymphatic; Malignant - descriptor; Massive Parallel Sequencing; Mediating; Medical; Megalencephaly; Morbidity - disease rate; Mosaicism; Mus; Mutate; Mutation; Operative Surgical Procedures; Other Genetics; PIK3CA gene; Paraffin Embedding; Parents; Pathogenesis; Pathologic; Pathologist; Patients; Pattern; Persons; Phenotype; Primary Health Care; Process; RNA; Reporter; Research; Research Personnel; Role; Sampling; Sensitivity and Specificity; Skeletal system; Skeleton; Skin; Somatic Mutation; Surgeon; Syndrome; Tamoxifen; Therapeutic; Tissue Sample; Tissues; Work; alternative treatment; bone loss; cDNA Library; cell type; cohort; embryonic stem cell; exome; experimental study; genetic analysis; inhibitor/antagonist; insight; malformation; mutant; novel; osteosarcoma; prevent; prospective; public health relevance; reproductive; sample collection; skeletal disorder; success; tool; transcriptome; transcriptome sequencing; treatment strategy

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.

描述（由申请人提供）：本申请重点关注大量患有影响骨骼疾病的儿童和成人，这些疾病是遗传性的，但不是遗传性的。这些疾病导致显着的发病率。 Gorham-Stout 病 (GSD)、全身淋巴管异常 (GLA)、骨囊肿和肿瘤就是这些疾病的例证。 该申请的共同研究人员开发了一种方法，通过将从患者受影响的组织和未受影响的组织中回收的DNA或RNA的大规模并行测序与计算算法相结合，识别这些类型疾病的遗传原因，该算法可检测体细胞嵌合突变，这些突变要么仅来自受影响的组织，要么与未受影响的组织相比，来自受影响组织的序列丰富。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的病理样本。我们验证了我们的方法，发现 PIK3CA 中的体细胞嵌合激活突变是导致 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、非遗传性疾病。 我们已经确定了几个患有 GSD、GLA 和骨肿瘤等疾病的明确特征的患者群体，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 名 GLA 患者、6 名 GSD 患者、20 名巨细胞瘤患者、38 名软骨母细胞瘤患者、8 名软骨粘液样纤维瘤患者的病变组织。在该提案的目标之一中，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，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的致病突变的计算算法。我们期望成功识别许多这些新疾病的致病突变。 我们提案的目标二是基于我们的发现，即当以体细胞嵌合形式存在时，PIK3CA 激活突变会导致图案缺陷、畸形和过度生长。我们有一只可以有条件地激活 p.1047H>R 突变的小鼠，我们在 CLOVES、KTS、FIL 和孤立性淋巴畸形患者中发现了这种突变，我们很快就会有一只可以有条件地激活然后灭活另一种致病性 Pik3ca 突变（p.420C>R）的小鼠。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发病机制，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进行基因分析的疾病的患者的表型联系起来，并且对于患有 PIK3CA 相关表型的患者，确定病变如何形成以及 PIK3CA 抑制是否是可行的治疗策略。