Rare & Mosaic Disorders Molecular Research

实验室的总体方法 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。在临床竞技场（研究HG 200388）中，我们实施了几项临床研究方案，以评估严重程度范围、畸形谱、多效性发育和过度生长疾病的自然史以及治疗研究。我们使用现代分子生物学的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，细胞和组织培养研究，以评估细胞生物学功能和基因产物的异常，以及人类遗传疾病的动物小鼠模型的创建和分析，现在我们已经开发了这些疾病的治疗方案。 过度生长综合征 基于我们先前在PIK 3CA相关纤维脂肪过度生长（Lindhurst et al，2012）和变形综合征（Lindhurst et al，2011）方面的成功，我们现在已经确定了治疗干预的靶点。这些发现表明，导致马赛克过度生长的突变也是癌症发病机制的主要贡献者。由于制药业对抗癌药物的大量研究，我们使用了两种这样的药物来治疗过度生长。 我们正在使用单细胞克隆成纤维细胞系与变形综合征患者测试治疗剂。通过根据细胞死亡和增殖试验滴定药物剂量，我们测量了这些药物对未来临床开发的潜在作用。这些临床前数据支持我们成功申请IND，使用该化合物治疗受影响的患者。 变形综合征建模 继我们在2011年发现这种疾病的原因后，我们已经成功地在小鼠中建立了这种疾病的模型。虽然Happle假说（非嵌合状态下致命突变的嵌合现象）与该疾病的所有公认特征完全一致，但在人类研究中无法证明这一点。为此，我们通过为p.Glu17Lys突变（影响所有已知患有这种疾病的患者的突变）创建条件性敲入等位基因，创建了Proteus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征一致的异常。我们正在计划对这个模型系统进行治疗实验。 我们还发现了血管重塑的异常。 镶嵌病的基因型-表型研究 我们已经开发了组织取样和培养方法，结合定制工程突变测定，以检测被认为受这种疾病影响的患者中这些基因中的5种已知突变，现在常规地将其作为患者筛查方法。这将使我们能够支持我们的临床研究项目（HG 200388）中的活动，以重新分类这些表型。对于该分析为阴性的任何患者，我们使用我们开创的患者内外显子组镶嵌比较方法将这些样品输入我们的下一代测序分析管道。我们还与NCI合作，开始了变形综合征肿瘤分子发病机制的研究。

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. We have also demonstrated abnormalities in vascular remodeling. 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. We have also initiated studies of the molecular pathogenesis of tumors in Proteus syndrome in collaboration with NCI.