Molecular Studies of Malformations

畸形的分子研究

基本信息

批准号：
10022456
负责人：
Leslie Biesecker
金额：
$ 168.41万
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/10022456
关键词：
AKT1 gene Affect Alleles Animal Model Animals Antineoplastic Agents Award Behavioral Biological Assay Biological Models Biological Process Biotechnology Cell Culture Techniques Cell Death Cell Proliferation Cells Cellular Assay Clinical Clinical Data Clinical Research Clinical Research Protocols Clinical Trials Clone Cells Congenital Abnormality Coupled Custom Data Development Dimensions Disease Disease model Dose Drug Industry Embryo Engineering Etiology Fibroblasts Future Gene Abnormality Genes Genetic Diseases Genetic Predisposition to Disease Genetic study Genomics Genotype High-Throughput Nucleotide Sequencing Human Human Genetics In Vitro International Knock-in Laboratories Malignant Neoplasms Measures Methods Microphthalmos Modeling Modernization Molecular Molecular Biology Molecular Genetics Mosaicism Mus Mutation Natural History Other Genetics PIK3CA gene Pathogenesis Patients Phase Phenotype Polyadenylation Polydactyly Proteins Proteus Proteus Syndrome Proto-Oncogene Proteins c-akt Protocols documentation Publishing Research Research Project Grants Sampling Severities Shapes Sirolimus Syndrome System Testing Therapeutic Therapeutic Agents Therapeutic Intervention Therapeutic Studies Tissue Sample Translational Research Treatment Protocols Variant Work analysis pipeline clinical development comparative congenital anomaly dosage exome experimental study feeding gene product inhibitor/antagonist malformation mouse model next generation sequencing novel novel therapeutics patient screening phase 2 study positional cloning pre-clinical recruit success targeted treatment therapeutic evaluation tissue culture tool

项目摘要

Overall Approach of the laboratory The laboratory uses a translational research approach to study human malformations and overgrowth disorders. In the clinical arena (study HG200388), 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 mouse models of human genetic disease, and now we have developed therapeutic protocols for several of these 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 have used two such agents 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 preclinical data supported our successful application for an IND to use this compound to treat affected patients. Modeling Proteus syndrome Following on our discovery of the cause of this disorder in 2011 we have been successful in modeling this disorder in mice. 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 created 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 shown that these animals have the predicted 100% embryonic lethality and a range of abnormalities consistent with the human syndrome. We are now planning therepeutic experiments for this model system. 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) 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 have determined the cause of a novel form of microphthalmia in the past year, and collaborated on two other international consortia gene identification studies.

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和过度生长障碍。在临床领域（研究 HG200388），我们实施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。这些包括高通量测序、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，以及人类遗传疾病的动物小鼠模型的创建和分析，现在我们有制定了针对其中几种疾病的治疗方案。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。由于制药行业对癌症药物的大量研究，我们使用了两种此类药物来治疗过度生长。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。这些临床前数据支持我们成功申请 IND，以使用该化合物治疗受影响的患者。变形杆菌综合症建模继 2011 年发现这种疾病的病因后，我们成功地在小鼠身上建立了这种疾病的模型。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，我们通过为 p.Glu17Lys 突变（影响所有已知患有这种疾病的突变）创建条件敲入等位基因，创建了变形杆菌综合征的小鼠模型。我们已经证明，这些动物具有预测的 100% 胚胎致死率和一系列与人类综合症一致的异常现象。我们现在正在计划对该模型系统进行重复实验。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) 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评估了大约 20 种独特的表型，并阐明了其中 5 种可能的遗传病因，这与其他群体的成功率非常相似。去年，我们确定了一种新型小眼症的病因，并合作开展了另外两项国际联盟基因鉴定研究。