Molecular Studies of Malformations

该实验室使用一种转化研究方法来研究人类畸形。在临床领域(研究HG200388-01)，我们运行了几个临床研究方案来评估严重程度、畸形的范围、多效性发育和过度生长障碍的自然病史以及治疗研究。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、位置克隆、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品异常的细胞和组织培养研究，以及人类遗传病动物模型(小鼠和斑马鱼)的建立和分析。 在过去的一年里，我们利用这些技术阐明了PIGA相关的中枢神经系统发育不良(Johnston等人，2012)和PIK3CA相关的纤维脂肪过度生长(Lindhurst等人，2012)的病因。我们已经进行了功能研究，以将突变与肌动蛋白的体外功能相关联，并进行了广泛的细胞培养和突变分析研究，以将AKT1突变的分布与皮肤表现的类型相关联(Lindhurst等人，在出版社) 马赛克病的基因-表型研究 在接下来的一年里，我们建议扩展我们的工作，将马赛克纤维脂肪过度生长的表现与我们在过去一年中发现的PIK3CA突变的类型和分布联系起来。我们正在开发组织采样和培养方法，并结合定制工程的突变分析，在被认为受到这种疾病影响的患者中检测这些基因中的5个已知突变。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。对于该分析为阴性的任何患者，我们建议使用我们首创的患者内外显子组马赛克比较方法进行下一代测序分析。我们还获得了一名具有典型变形杆菌综合征临床特征的患者的大量尸检样本，并将对这些样本进行组织学突变关联，以了解患者的分布范围及其与突变负荷的潜在相关性。 新生殖系表型的外显子组分析 多年来，我们在描绘与GLI3相关疾病相关的突变谱方面取得了成功。然而，尽管取得了这些成功，但仍有相当数量的患者的已知突变为阴性。我们建议使用外显子组测序来分析15名此类患者的队列，以阐明这些疾病的病因。 建立Proteus综合征模型 在2011年我们发现了这种疾病的原因之后，我们一直在寻求一种策略来模拟这种疾病。虽然HApple假说(在非马赛克状态下的马赛克致死突变)与这种疾病的所有公认特征完全一致，但在人类研究中不可能证明这一点。此外，虽然这种激活的突变提供了一个非常诱人的治疗靶点，但我们进行临床前研究的能力再次受到限制。为此，我们致力于通过创建P.Glu17Lys突变的条件性敲入等位基因来创建变形杆菌综合征的小鼠模型，该突变会影响所有已知的这种疾病患者。我们在这个项目上取得了很好的进展，完成了广泛的基因工程来制造和验证该结构。该构建物已被注射到动物体内，目前正在进行筛选。我们还使用来自变形杆菌综合征患者的单细胞克隆成纤维细胞株来测试治疗药物。通过滴定药物的剂量与细胞死亡和增殖的分析，我们可以衡量这些药物在未来临床开发中的潜在效果。

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). Using these techniques in the past year we have elucidated the etiology of PIGA-related CNS dysplasia (Johnston et al, 2012) and PIK3CA-related fibroadipose overgrowth (Lindhurst et al, 2012). We have performed functional studies to correlate mutations with in vitro functioning of actin and we have performed extensive cell culture and mutation analysis studies to correlate the distribution of AKT1 mutations with the type of skin manifestations (Lindhurst et al, in Press) Genotype-Phenotype studies in mosaic disorders In the coming year we propose to extend our work correlating the manifestations of mosaic fibroadipose overgrowth with the type and distribution of mutations in PIK3CA, which we discovered this past year. We are developing 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. 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 then propose to move on to next generation sequencing analysis using the intrapatient exome mosaic comparison approach that we have pioneered. We have also come into possession of a large number of autopsy samples from a patient with classical clinical features of Proteus syndrome and will perform a histologic-mutational correlation with these samples to understand the range of distribution in a patient and the potential correlation with mutation load. Exome analysis of novel germline phenotypes We have had success over a number of years in delineating the spectrum of mutations associated with GLI3-related disorders. Yet, in spite of these successes, a substantial number of patients have been negative for known mutations. We propose to analyze a cohort of 15 such patients using exome sequencing to elucidate the etiology of these 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. As well, while this activating mutation provides a very tempting therapeutic target, again we are limited in our ability to do preclinical 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 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. We are also 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 can measure the potential effect of these agents for future clinical development.