Molecular Studies of Malformations

畸形的分子研究

基本信息

批准号：
8948372
负责人：
Leslie Biesecker
金额：
$ 152.46万
依托单位：
NATIONAL HUMAN GENOME RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8948372
关键词：
AKT1 gene Affect Alleles Animal Model Animals Antineoplastic Agents Autopsy Behavioral Biological Assay Cell Death Cell Proliferation Cells Clinic Clinical Clinical Data Clinical Research Clinical Research Protocols Clone Cells Congenital Abnormality Coupled Custom Data Development Dimensions Disease Disease model Drug Industry Engineering Etiology Family Fibroblasts Future Genes Genetic Engineering Genetic Predisposition to Disease Genets Genomics Genotype Hereditary Disease High-Throughput Nucleotide Sequencing Histology Human Human Genetics In Vitro International Knock-in Mouse Laboratories Malignant Neoplasms Measures Methods Modeling Molecular Molecular Biology Molecular Genetics Mosaicism Mus Mutation Natural History Other Genetics PIK3CA gene Pathogenesis Pathogenicity Patients Phenotype Polydactyly Proteins Proteus Syndrome Reporting Research Research Project Grants Resources Sampling Sequence Analysis Severities Shapes Skin Manifestations Syndrome System Testing Therapeutic Therapeutic Agents Therapeutic Intervention Therapeutic Studies Therapy Clinical Trials Tissue Sample Translational Research Work Zebrafish congenital anomaly diagnosis standard dosage exome feeding malformation meetings mouse model next generation sequencing novel novel therapeutics positional cloning pre-clinical programs research study screening success tissue culture tissue/cell culture tool

项目摘要

Overall Approach of the laboratory The laboratory uses a translational research approach to study human malformations. In the clinical arena (study HG200388-01), we operate several clinical research protocols to assess the range of severity, spectrum of malformations, natural history of pleiotropic developmental and overgrowth disorders and therapeutic studies. We use the tools of modern molecular biology to determine the molecular pathogenesis of these disorders. These include high throughput sequencing, positional cloning, microarray expression and microarray CGH analysis, cell and tissue culture studies to assess cell biologic functions and abnormalities of gene products, and the creation and analysis of animal models of human genetic disease (mouse and zebrafish), and now we are moving into therapeutic approaches to the diseases. Overgrowth syndromes Building on our prior successes with PIK3CA-related fibroadipose overgrowth (Lindhurst et al, 2012) and Proteus syndrome (Lindhurst et al, 2011), we have now identified targets for therapeutic intervention. These discoveries have shown that the mutations that cause mosaic overgrowth also are major contributors to the pathogenesis of cancer. Because of the intense work in the pharmaceutical industry on cancer drugs, we can exploit those resources and repurpose them for therapeutics of overgrowth. We are using single-cell cloned fibroblast lines from patients with Proteus syndrome to test therapeutic agents. By titrating dosage of agents against assays of cell death and proliferation we have measured the potential effect of these agents for future clinical development. These experiments are being completed and have been submitted for presentation at the Am Soc Hum Genet meeting in 2014. We have also successfully correlated the distribution of AKT1 mutations with the type of skin manifestations (Lindhurst et al, 2014) and we are currently working on a detailed mutational analysis of an autopsy case of Proteus syndrome where we have correlated mutational load with histology in more than 50 tissue samples, by far the most detailed study of its kind in humans. We have also convened an international group to set clinical standards of diagnosis and workup for patients with PIK3CA-related overgrowth and have founded an international consortium to develop cooperative trials for pharmacologic treatment of this family of disorders. Modeling Proteus syndrome Following on our discovery of the cause of this disorder in 2011 we have been pursuing a strategy to model this disorder. While the Happle hypothesis (mosaicism for a mutation lethal in the non-mosaic state) is completely consistent with all recognized features of the disorder, it is impossible to prove this in human studies. To that end, we have undertaken efforts to create a mouse model of Proteus syndrome by creating a conditional knock-in allele for the p.Glu17Lys mutation (the mutation that affects all known patients with this disorder). We have made excellent progress on this project with extensive genetic engineering accomplished to make and validate the construct. The construct has been injected into animals and is currently being screened. Genotype-Phenotype studies in mosaic disorders We have developed tissue sampling and culture methods coupled with custom-engineered mutation assays to detect the 5 known mutations in these genes in patients thought to be affected with this disorder and now routinely perform this as a patient screening method. This will allow us to support the activities in our clinical research project (HG200388-01) to reclassify these phenotypes. For any patients in whom this analysis is negative, we feed these samples into our next generation sequencing analysis pipeline using the intrapatient exome mosaic comparison approach that we have pioneered. Exome analysis of novel germline phenotypes In this past year we have used exome analysis to evaluate about 20 distinctive phenotypes, with the elucidation of probable genetic etiologies for 5 of these (which is quite similar to the success rates for other groups. We are currently pursuing two of these with functional studies to assess the pathogenicity of the mutations we have identified.

实验室总体思路该实验室采用转化研究方法来研究人类畸形。在临床领域（研究 HG200388-01），我们实施了多项临床研究方案来评估严重程度、畸形范围、多效性发育和过度生长障碍的自然史以及治疗研究。我们使用现代分子生物学工具来确定这些疾病的分子发病机制。其中包括高通量测序、定位克隆、微阵列表达和微阵列 CGH 分析、评估细胞生物学功能和基因产物异常的细胞和组织培养研究，以及人类遗传疾病动物模型（小鼠和斑马鱼）的创建和分析，现在我们正在研究这些疾病的治疗方法。过度生长综合症基于我们之前在 PIK3CA 相关纤维脂肪过度生长（Lindhurst 等人，2012）和 Proteus 综合征（Lindhurst 等人，2011）方面取得的成功，我们现在已经确定了治疗干预的目标。这些发现表明，导致嵌合体过度生长的突变也是癌症发病机制的主要贡献者。由于制药行业在癌症药物方面开展了大量工作，我们可以利用这些资源并将其重新用于过度生长的治疗。我们正在使用变形杆菌综合征患者的单细胞克隆成纤维细胞系来测试治疗药物。通过根据细胞死亡和增殖的测定滴定药物剂量，我们测量了这些药物对未来临床开发的潜在影响。这些实验正在完成，并已提交到 2014 年 Am Soc Hum Genet 会议上进行演示。我们还成功地将 AKT1 突变的分布与皮肤表现类型相关联（Lindhurst 等，2014），并且我们目前正在对 Proteus 综合征的尸检病例进行详细的突变分析，其中我们将突变负荷与组织学联系起来更多超过 50 个组织样本，这是迄今为止对人类进行的最详细的同类研究。我们还召集了一个国际小组来制定 PIK3CA 相关过度生长患者的诊断和检查临床标准，并成立了一个国际联盟来开发针对该家族疾病的药物治疗合作试验。变形杆菌综合症建模继 2011 年发现这种疾病的病因后，我们一直在寻求一种策略来模拟这种疾病。虽然哈普尔假说（非马赛克状态下致命突变的马赛克现象）与该疾病的所有公认特征完全一致，但不可能在人类研究中证明这一点。为此，我们通过创建 p.Glu17Lys 突变（影响所有已知患有这种疾病的突变）的条件敲入等位基因，努力创建变形杆菌综合征的小鼠模型。我们在这个项目上取得了巨大的进展，完成了广泛的基因工程来制造和验证该结构。该构建体已被注射到动物体内，目前正在进行筛选。嵌合病的基因型-表型研究我们开发了组织取样和培养方法，并结合定制工程突变测定，以检测被认为患有这种疾病的患者中这些基因的 5 种已知突变，现在将其作为患者筛查方法常规执行。这将使我们能够支持我们的临床研究项目 (HG200388-01) 中的活动，以重新分类这些表型。对于任何分析结果为阴性的患者，我们使用我们首创的患者内外显子组嵌合比较方法将这些样本输入我们的下一代测序分析流程。新种系表型的外显子组分析在过去的一年里，我们使用外显子组分析来评估约 20 种独特的表型，并阐明了其中 5 种的可能遗传病因（这与其他群体的成功率非常相似。我们目前正在通过功能研究来研究其中两种表型）评估我们已确定的突变的致病性。