Molecular Studies of Malformations

该实验室使用转化研究方法来研究人类畸形。在临床竞技场（研究HG 200388 -01）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、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。 在过去的一年中，我们使用这些技术阐明了PIGA相关CNS发育不良（约翰斯顿等人，2012）和PIK 3CA相关纤维脂肪过度生长（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，并且我们已经进行了广泛的细胞培养和突变分析研究，以将AKT 1突变的分布与皮肤表现的类型相关联（Lindhurst等人，出版中）。 镶嵌病的基因型-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去年发现的PIK 3CA突变的类型和分布相关联。我们正在开发组织取样和培养方法，并结合定制的突变检测方法，以检测被认为患有这种疾病的患者中这些基因中的5种已知突变。这将使我们能够支持我们的临床研究项目（HG 200388 -01）中的活动，以重新分类这些表型。对于这种分析是阴性的任何患者，我们建议使用我们开创的患者内外显子组镶嵌比较方法进行下一代测序分析。我们还从一名具有变形综合征典型临床特征的患者中获得了大量尸检样本，并将对这些样本进行组织学-突变相关性分析，以了解患者中的分布范围以及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，我们已经成功地描绘了与GLI 3相关疾病相关的突变谱。然而，尽管取得了这些成功，但仍有相当数量的患者对已知的突变呈阴性。我们建议使用外显子组测序分析15例此类患者的队列，以阐明这些疾病的病因。 变形综合征建模 在2011年我们发现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（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。同样，虽然这种激活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仍然有限。为此，我们已经进行了努力，通过创建p.Glu17Lys突变的条件性敲入等位基因来创建Proteus综合征的小鼠模型，该突变影响所有已知的患有这种疾病的患者。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构建体。该构建体已被注射到动物体内，目前正在筛选中。我们还使用从变形综合征患者身上克隆的单细胞成纤维细胞系来测试治疗剂。通过根据细胞死亡和增殖试验滴定药物剂量，我们可以测量这些药物对未来临床开发的潜在作用。

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.